Method of conditioning a hybrid synthetic tubular structure to yeild a functional human hybrid hemodialysis access graft

ABSTRACT

A method of yielding a functional human hybrid hemodialysis access graft is provided. The method includes conditioning a hybrid synthetic tubular structure having stem cells and/or endothelial cells on at least one surface to yield the functional human hybrid hemodialysis access graft. Specifically, the method includes placing the hybrid synthetic tubular structure in a system capable of producing three dimensional dynamic conditions for a sufficient time to yield said functional human hybrid hemodialysis access graft.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of PCT/US2006/045715, filed Nov. 30,2006 and a Continuation-in-Part of U.S. application Ser. Nos.11/440,152, filed May 25, 2006; 11/440,156, filed May 25, 2006, filedMay 25, 2006; 11/440,155, filed May 25, 2006; 11/440,091, filed May 25,2006; 11/440,158, filed May 25, 2006, which in turn are Continuations ofU.S. application Ser. No. 09/973,433, filed Oct. 8, 2001, now U.S. Pat.No. 7,063,942 and International Application No. PCT/US2001/042576, filedOct. 9, 2001, now Publication No. WO 2002/032224 A1, which claim thebenefit of U.S. Provisional Application No. 60/239,015, filed Oct. 6,2000. The entire disclosure of the prior applications are considered asbeing part of the disclosure of the accompanying application and arehereby incorporated by reference therein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a method for making a human hybrid hemodialysisaccess graft and, more particularly, a method of conditioning a hybridsynthetic tubular structure having stem cells and/or endothelial cellson at least one surface to yield a functional human hybrid hemodialysisaccess graft.

2. Background of the Related Art

Hemodynamics plays an obligate role on the function and phenotype ofvascular cells (i.e. endothelial cells, smooth muscle cells,fibroblasts, etc.) and tissues in the cardiovascular system duringdisease and healthy states. Cardiovascular disease is the leading causeof death in North America, Europe and the developing world, withcoronary heart disease and atherosclerosis being amongst the mostprominent cardiovascular diseases. Atherosclerosis is a disorder inwhich the coronary arteries become clogged by the build up of plaquealong the interior walls of the arteries, leading to decreased bloodflow which can in turn cause hypertension, ischemias, strokes and,potentially, death. Associated systemic risk factors includehypertension, diabetes mellitus, and hyperlipidemia, among otherfactors.

Atherosclerosis and other cardiovascular diseases, such as peripheralarterial disease (PAD), occur regularly and predictably at sites ofcomplex hemodynamic behavior and, consequently, motivates furtherinvestigation into the role of hemodynamics in cardiovascular diseases.For example, atherosclerosis has been shown to occur in sites of complexhemodynamic behavior. Surgical intervention is often employed to treatit, and may include insertion of a balloon catherter to clean out theplaquie, and insertion of a stent within the vessel to enable it toremain open, or may include multiple bypasses of the clogged vessels.Bypass surgery involves the removal of a section of vein from thepatient's lower leg, and its transplant into the appropriate cardiacblood vessels so that blood flows through the transplanted vein and thusbypasses the clogged vessels. A major problem associated with bypasssurgery is the patency of the vessels to be used in the bypass. Thebypass vessels are prone to failure, which may occur within a shortperiod of time after bypass surgery, or after a period of several years.Hemodynamic forces have been implicated as a major factor contributingto the failure of the bypass vessels.

Hemodynamic forces, which are forces generated by irregular flow, and inparticular, by the (sometimes irregular) flow of blood, are known tohave numerous influences on blood vessels, including, but not limited toeffects on blood vessel cell structure, pathology, function, anddevelopment. In the specific example of blood vessel structure andpathology, the vascular cells lining all blood vessels, endothelialcells (ECs), are important sensors and transducers of two of the majorhemodynamic forces to which they are exposed. These forces include wallshear stress (“WSS”), which is the fluid frictional force per unit ofsurface area, and hoop stress, which is driven by the circumferentialstrain (“CS”) of pressure changes. Wall shear stress acts along theblood vessel's longitudinal axis, while circumferential strain isassociated with the deformation of the elastic artery wall (i.e.,changes in the diameter of the vessel) in response to oscillation orvariation in vascular pressure. Wave reflections in the circulation andthe inertial effects of blood flow cause a phase difference, the stressphase angle (“SPA”), between CS and WSS. The SPA vanes significantlythroughout the circulation, and is most negative in disease pronelocations, such as the outer walls of a blood vessel bifurcation such asthe carotid sinus and the coronary arteries. Hemodynamic forces havebeen shown to dramatically alter endothelial cell function and phenotype(i.e., higher shear stress [low SPA] is associated with anatheroprotective gene expression profile, and a low shear stress [largeSPA] is associated with an atherogenic gene expression profile).

ECs can influence vasoactivity and cause vessels to contract or dilatedepending on the blood flow (shear stress) and pressure (causing stretchor CS), and thus are one component which is critical to blood pressureregulation among the many important factors which influence and/or aredependent on the hemodynamics. ECs are just one type of cell which isdirectly influenced by hemodynamics. Numerous other cell types may alsodirectly or indirectly influenced by hemodynamics and mechanical forces.

As discussed above, hemodynamic forces have been shown to dramaticallyalter endothelial function and phenotype. For example, the coronaryarteries are the most disease prone arteries in the circulation and havethe most extreme SPA in the circulatory system, typically having alarge, negative value, yet do not have a particularly low shear stressmagnitude, thus suggesting that complex hemodynamic factors that includethe SPA are important in cardiovascular function and pathology.Accordingly, there is a great need to study vascular biology in acomplete, integrated, and controlled hemodynamic environment, preferablyin 3-dimensions. However, to date, detailed knowledge of thesimultaneous, combined influence of the time varying patterns of WSS andCS on EC biological response has not been technologically feasible.

More specifically, existing systems have focused on the individualeffects of either WSS or strain on ECs separately. The most common WSSsystems use a 2-dimensional stiff surface, such as, for example, a glassslide, for the EC culture on the wall of a parallel plate flow chamber,or a cone-and-plate type chamber, to simulate wall shear stress alone,which is only one hemodynamic condition. In such a system, the WSS mustusually remain steady due to difficulties in simulating pulsatile flow,and strain or stretch effects must be omitted. Further, cyclic strainingdevices can only generate strain by stretching cells on a compliantmembrane, without flow, and typically only in 2-dimensions. Both typesof systems are obviously limited in the fidelity with which they cansimulate a true, complete hemodynamic environment.

To address the need for simultaneous pulsatile strain and shear stress,a silicone tube coated with ECs was introduced. However, simulatorsusing these tubes could only achieve phase angles (SPA) of about −90degrees, if any, which is inadequate for simulating coronary arteries(SPA>−180 or −250 degrees), the most disease prone vessels in thecirculation, or other regions of the circulation such as peripheralcirculation, carotid, renal, organ hemodynamics, or head and brainhemodynamics, to name a few. A more complete physiologic environmentwhich provides time-varying uniform cyclic CS and pulsatile WSS in a3-dimensional configuration over a complete range of SPA is sill needed.

Substantially all past research and development has focused only onobvious, one-dimensional blood flow or shear stress hemodynamic forcecharacteristics, even though, based on physics, mathematics, andexperimentation, there are clearly a multitude of dimensions associatedwith the with many simultaneous hemodynamic forces present in vivo, suchas pressure and strain. Physiologic environments are highly dynamic andnonlinear, the cardiovascular system is certainly no exception. There isa need to preserve 3-dimensional vascular geometry while simultaneouslyand independently controlling hemodynamic forces such as, for example,pressure, flow, and stretch, as well as many other parameters andforces) in a cell and tissue culture environment in order to more fullyand more accurately recapitulate in vivo hemodynamic environments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described in detail with reference to thefollowing drawings in which like reference numerals refer to likeelements, wherein:

FIG. 1A is a schematic view of a system for recreating a hemodynamicenvironment in accordance with an embodiment of the invention;

FIG. 1B is a schematic view of a reservoir for use with the system shownin FIG. 1A;

FIGS. 2A-2E are schematic views of systems for recreating a hemodynamicenvironment in accordance with embodiments of the invention;

FIGS. 3A-3D are schematic views of systems for recreating a hemodynamicenvironment in accordance with embodiments of the invention;

FIGS. 4A-4D are schematic views of a chamber which may be applied withany of the systems shown in FIGS. 2A-2E and 3A-3D;

FIGS. 5A-5C illustrate specimen shapes which may be applied with any ofthe systems shown in FIGS. 2A-2E and 3A-3D;

FIGS. 6A-6E illustrate exemplary chamber(s)s with specimen(s) mountedtherein which may be applied with any of the systems shown in FIGS.2A-2E and 3A-3D;

FIGS. 7A-7D illustrate an exemplary mounting system which may be appliedwith any of the systems shown in FIGS. 2A-2E and 3A-3D;

FIGS. 8A-8E illustrate a coupling system which may be applied with anyof the systems shown in FIGS. 2A-2E and 3A-3D;

FIGS. 9A-9B are flowcharts illustrating operation of the systems shownin FIGS. 2A-2E and 3A-3D; and

FIGS. 10A-10H are graphs of pressure, diameter and flow rate conditionsgenerated by the systems shown in FIGS. 2A-2E and 3A-3D.

FIG. 11 shows a side view of a specimen in accordance with an embodimentof the invention;

FIG. 12 shows examples of cross-sections of tubular structures accordingto various embodiments of the invention;

FIG. 13A shows several examples for the measurement of the parameterD(t);

FIG. 13B is a schematic cross sectional view of an example of amulti-layer tubular structure;

FIG. 14 shows examples of tubular structures;

FIG. 15 shows multiple regions in an exemplary tubular structure wheredynamic conditions can be linked to global dynamic conditions measuredat the input and the output, respectively;

FIG. 16 shows an alternative block diagram of a system according toanother embodiment of the invention;

FIGS. 17A and 17B show examples of various forms or types of dynamicconditions;

FIG. 18 shows examples of classes of dynamic conditions that can besimulated according to various embodiments of the invention;

FIG. 19 shows a block diagram of a controller according to an embodimentof the invention;

FIG. 20 shows a block diagram of a translator according to an embodimentof the invention;

FIG. 21 shows an exemplary physiological coronary flow;

FIG. 22 shows an exemplary pressure/flow loop subsystem in accordancewith an embodiment of the invention;

FIGS. 23 a-23 d are diagrams that show various stages of a plurality ofpumps;

FIG. 24 shows a plurality of states during one cycle of operation;

FIGS. 25A-25C exemplary dynamic conditions relating relative phases ofpressure and flow;

FIG. 26 shows a schematic diagram of an exemplary pump;

FIG. 27 shows a motor controller in accordance with an embodiment of theinvention;

FIG. 28 shows a controller having a processor coupled to a pumpcontroller in accordance with an embodiment of the invention;

FIG. 29 shows an exemplary flowchart to determine control signalscorresponding to dynamic conditions and/or input information accordingto an embodiment of the invention;

FIG. 30 shows variations of a first order harmonic ω₁(t) of a dynamicvariable g(t) in accordance with an embodiment of the invention;

FIGS. 31A-31B shows an example of the variations in time ofcharacteristics of three harmonics of a dynamic condition;

FIG. 32 shows variations of the _(i)th harmonic amplitude of a dynamiccondition in accordance with embodiments of the invention;

FIGS. 33A and 33B show representative frequencies and amplitudes fordifferent physiological experiences, respectively;

FIG. 34 shows evolution of a plurality of types of dynamic conditionsfor a physiological experience;

FIG. 35A shows a flowchart of a method for creating physiologicalexperiences for a patient with a particular patient history;

FIG. 35B lists examples of physiological experiences;

FIGS. 36 and 37 show block diagrams of systems with controller and apressure/flow subsystem that generate a flow loop of fluid according toembodiments of the invention;

FIGS. 38 and 39 show block diagrams of systems with pressure/flow loopsubsystems according to embodiments of the invention;

FIG. 40 shows system with sensors according to embodiments of theinvention;

FIGS. 41A-41C show exemplary electrode configurations for measuringdynamic conditions;

FIGS. 42A-42B and 43A-43B shows examples of exemplary sensorscommunicatively coupled to transmit, receive, transmit and receive,detect and forward data used as feedback;

FIGS. 44A-44B show exemplary embodiments of a prove sensor according toan embodiment of the invention.

FIGS. 45A-45C and 46 show exemplary tubular structures in accordancewith embodiments of the invention;

FIGS. 47A-47G show exemplary tubular structures illustrating exemplarysecond order dynamic conditions in accordance with embodiments of theinvention;

FIG. 48 show a flowchart of a process of matching second order dynamicconditions to a selected target according to an embodiment of theinvention;

FIG. 49 shows a flowchart of a process for combining biologic andnon-biological materials according to an embodiment of the invention;and

FIG. 50 shows NO levels for cells experiencing different dynamicconditions produced by an embodiment of a system according to theinvention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Any reference in this specification to “one embodiment,” “anembodiment,” “example embodiment,” “embodiments,” etc., means that aparticular feature, structure, or characteristic described in connectionwith the embodiment is included in at least one embodiment of theinvention. The appearances of such phrases in various places in thespecification are not necessarily all referring to the same embodiment.Further, when a particular feature, structure, or characteristic isdescribed in connection with any embodiment, it is submitted that it iswithin the purview of one skilled in the art to effect such feature,structure, or characteristic in connection with other ones of theembodiments.

A hemodynamic simulation system in accordance with embodiments of theinvention as embodied and broadly described herein overcomes currenttechnological limitations in biomedical research and, particularly, invascular research are overcome by physically reproducing both normal anddiseased physiologic states in a controlled environment. A precise andcomplete physiologic environment is achieved via control of salientdynamic conditions such as, for example, pressure, flow, and diameter,that consequently control the predominant dynamic forces, WSS and CS.This is achieved through independent control of these dynamicconditions, thus allowing for independent control over a variety ofdynamic parameters and forces such as the magnitude and phase of thepulsatile WSS and CS at a wide range of SPA. The system provides for therecreation of real dynamic patterns, complex and simple, while alsomeeting the stringent requirements for sterility and minimal mediavolume critical in cell and tissue culture systems.

The system neatly integrates engineering and biological principles byimposing a realistic, time varying mechanical environment on a testspecimen, such as, for example, living vascular cells, to provide amodel of normal and diseased cardiovascular function to help guide manyareas such as future therapeutic strategies, stem cell therapy, cell andtissue regeneration or engineering, genetic or pharmacologic. Theindependent control of pulsatile flow and pulsatile pressure to providefor independent control over WSS, CS and pressure is a significantbreakthrough which, at first, seems paradoxical. That is, classically,pressure and flow are coupled. However, in a dynamic oscillatory orsinusoidal environment such as is present in this system, flow andpressure can be independently controlled in a variety of ways to achievethe desired result. [DRM—Where do we describe the possible fluidcomponent compositions??]

FIG. 1A is a schematic view of a system for reproducing a hemodynamicenvironment and, more particularly, a schematic view of a flow loop ofsuch a system, in accordance with one embodiment of the invention asbroadly described herein. In this system 1, flow of fluid and/or mediais initiated by a steady flow system 30 and introduced into a flow loop,where it passes into a specimen unit 10. An individual or multiplespecimen 12 may be positioned in the specimen unit 10 by a mountingsystem 80. The single/multiple specimen 12 are exposed to fluid and/ormedia carried by the fluid, as well as to the dynamic environmentproduced by the system 1. The specimen unit 10 may be coupled, andpreferably detachably coupled, to the flow loop by a coupling system300.

Dynamic pressure and flow conditions within the specimen unit 10 may begenerated and maintained by a pressure/flow control system 200, whichacts on the fluid traversing through the flow. Fluid may besubstantially continuously recirculated through the flow loop for arequired amount of time/cycles, or based on another such controllingparameter which would govern the flow through the flow loop. In otherembodiments, a predetermined amount of fluid/media may be introducedinto the flow loop and held in the specimen unit 10 for a predeterminedamount of time/cycles, or other such controlling parameter, as thepressure/flow control system 200 generates the required conditions inthe specimen unit 10.

The action of the steady flow system 30 and the pressure/flow controlsystem 200 may be controlled by a control system 70. The control system70 may also receive data related to various parameters from varioussensors positioned throughout various portions of the system 100, suchas, for example, the specimen unit 10, the steady flow system 30, thepressure/flow control system 200, and other locations along the flowloop. In certain embodiments, the control system 70 provides for dynamiccontrol of the system 1 based on feedback provided by a variety ofsensing/detection systems (not shown in detail in FIG. 1A). Inalternative embodiments, the control system 70 may simply operate thesystem 100 in accordance with a previously stored algorithm based onconditions desired in the specimen unit 10 and/or throughout the flowloop, without feedback.

FIG. 1B is a schematic view of a reservoir 20 that may be optionallyused in the steady flow system 30 shown in FIG. 1A. The reservoir 20 mayhold fluid for initial and re-circulation, and may allow media to beintroduced into or siphoned from the flow loop. That is, as fluid/mediais returned to the reservoir 20, a portion, or all of the fluid/mediamay be redirected, or siphoned off and not recirculated. For thispurpose, the reservoir 20 may be partitioned into inflow 20 a andoutflow 20 b portions, or the siphoned fluid may be diverted to aholding tank or other such vessel or flow system (not shown).

The reservoir 20 may further include a sampling port 21 which samplesincoming fluid before recirculation and/or diversion to the outflowportion 20 b or a holding tank. The sampling port 21 may be adapted todivert incoming fluid based on, for example, its measurement ofparameters such as, for example, concentration of media components,contamination levels, circulation time/cycles and the like. Likewise,the incoming portion 20 a of the reservoir 20 may include an inflow port22 to allow for the introduction of additional fluid and/or media asrequired, and may include sensors 23 linked to the control system 70which continuously monitor levels/quantity of such fluid/media as it isintroduced into the flow loop.

The reservoir 20 may also include a port to atmosphere (not shown),preferably with a sterile filter to preclude contamination from theatmosphere. Additionally other cell or tissue types may be positionedthroughout the system, such as, for example, near the reservoir 20 or aport thereof. For example, a chamber (not shown) containing hepatocytesmay be positioned in the flow loop so as to be exposed to the fluid inthe flow loop, as well as to at least some of the dynamic conditions inthe flow loop, if desired. This type of exemplary setup can be used toprovide other useful data such as, for example, drug metabolism data.

The positioning and interconnection of the components of the system 1shown in FIG. 1A is merely exemplary in nature, and intended simply toillustrate the presence of these components and their respectivefunctions within the system 1. Thus, for example, although the steadyflow system 30 shown in FIG. 1 is positioned adjacent the reservoir 20and the pressure/flow control system 200, followed in the flow loop by aportion of the coupling system 300, it is well understood that thesteady flow system 30 may include various components positionedthroughout the system 1 to provide the capabilities required of thesteady flow system 30. Likewise, although the pressure/flow controlsystem 200 is shown simply on an ingress side of the specimen unit 10,it is well understood that the pressure/flow control system 200 mayinclude various components positioned throughout the system 1 to fulfillthe requirements of the pressure/flow control system 200. Such reasoningapplies to the remaining components of the system 1, including thecoupling system 300, mounting system 80, control system 70, and specimenunit 10, as will be better understood from the following discussion.

FIG. 2A is a schematic view of an exemplary system 1000 for reproducinga hemodynamic environment, in accordance with one embodiment of theinvention as broadly described herein. Although the specimen unit 10shown in FIG. 2A includes a chamber 11, which forms an enclosure for asingle specimen 12, the system 1000 may also include a single chamber 11housing a plurality of specimens 12, a plurality of chambers 11 eachhousing a single specimen 12, a plurality of chambers 11 each housing aplurality of specimens 12, and a plurality of chambers 11, some housinga single specimen 12, and some housing a plurality of specimens 12, aswill be further described below in connection with FIGS. 6A-6C. Further,as shown in FIGS. 6D-6E, the system 1000 may also include anindividual/multiple specimen 12 not surrounded by any type of enclosureor chamber 11. Instead, an individual/multiple specimen 12 may bealigned directly with the flow loop.

In alternative embodiments, the chamber 11 may be jacketed 15, as shown,for example, in FIG. 4B, thereby enabling circulation of a cooled orheated fluid through the chamber 11 and specimen 12, in order tomaintain the temperature required by the specimen 12 and an associatedtrial. Alternatively, the chamber 11 may be immersed in a water bath 16at an appropriate temperature, as shown, for example, in FIG. 4C, or mayinclude a conditioned circulation path 17, as shown, for example, inFIG. 4D to achieve the desired temperature control effects.

The system 1000 may generally be run at a temperature of approximately37 degrees Centigrade, but can be operated at temperatures ranging fromapproximately 20 degrees Centrigrade to approximately 50 degreesCentigrade, or whatever temperature may be required for a particulartrial.

**The specimen 12 may take many forms. In certain embodiments, thespecimen 12 may be a substantially tubular type, compliant structuremade of materials such as, for example, silicone, collagen, PTFE,fibrin, and other such appropriate materials, which is lined with avariety of cellular compounds and/or cells, such as, for example,endothelial cells or stem cells on a fibronectin matrix used to simulatea vessel wall a non-rigid tube that contains mammalian cells, a bloodvessel excised from a mammal, or other biocompatible substratecontaining cells or onto which cells can be grown or attached thereto.In other embodiments, the specimen 12 may be a portion of an actualvessel (ex vivo), such as, for example artery or vein, which is to besubjected to the hemodynamic environment produced by the system 1000.Likewise, while the specimen 12 discussed herein are, simply for ease ofdiscussion, substantially tubular, the specimen 12 may also have anirregular form to more accurately represent an actual physiologicalcondition or environment, such as, for example, a bifurcation a curve,physiologic vascular segment, or changes in cross section to reproduce aconstriction present in an actual vessel. Samples of some specimen 12which have such irregular forms are shown in FIGS. 5A-5C.

The specimen 12 may include various entities, such as, for example,different cell types. These may be cells other than vascular cells whichmay be attached or integrated in the specimen 12, or which may benon-attached and circulating, such as, for example, immune cells, suchas, for example, leukocytes, monocytes, and the like, stem cells, suchas, for example, adult, embryonic, progenitor, and the like, cancercells, red blood cells, platelets, and other such cell types. Otherorgan cells such as, for example, hepatocytes for liver toxicityassessment or adsorption distribution metabolism excretion (ADME)examination, may also be incorporated into the system for activitiessuch as, for example, testing and screening purposes. Similarly,numerous different components may be added to the media to simulatedifferent conditions, including, but not limited to, cholesterol forhyperchloesterolimia, growth factors for growth and development, calciumfor vulnerable plaque and lesion formation, and other such components.

In the system 1000 shown in FIG. 2A, the steady flow system 30 includesa reservoir 20 and a steady flow pump 30 a. Fluid which is to beintroduced into the specimen unit 10 may be drawn out of the reservoir20 by the steady flow pump 30 a which initiates and maintains asubstantially constant, substantially uniform flow of fluid from thereservoir 20 into the flow loop. Other types of pumps or componentswhich may be used to initiate and maintain such a steady flow may alsobe appropriate. The steady flow pump 30 a shown in FIG. 2A is disposedbetween the reservoir 20 and the specimen unit 10, at a positionupstream from an ingress 10 a into the specimen unit 10. However, thesteady flow pump 30 a may also be disposed at other positions within thesystem 1000, based on the type of component(s) used to generate thesteady flow from the reservoir 20, as well as the placement of othercomponents of the system 1000.

The desired test environment may be developed and maintained within thespecimen unit 10 by the pressure/flow control system 200. In the system1000 shown in FIG. 2A, the pressure/flow control system 200 may includea first pressure/flow control 40 positioned upstream of the ingress 10 ainto the specimen unit 10 and a second pressure/flow control 50positioned downstream of an egress 10 b from the specimen unit 10. Inalternative embodiments, the system 1000 may also include a thirdpressure/flow control 60 which further controls an internal pressureand/or flow within the specimen unit 10, and/or an external pressure.The first, second and third pressure/flow controls 40, 50 and 60 may becombined as necessary during operation of the system 1000, depending onwhich conditions are to be reproduced in the specimen unit 10 and whichproperties are to be monitored/studied during a particular trial. Forexample, the third pressure/flow control 60 may not be required in somesituations, such as, for example, when a specimen 12 is aligned directlywith the flow loop, without a chamber 11 surrounding the specimen, asshown in FIGS. 6D-6E, or when there is a chamber 11 in use but all therequired trial conditions can be reproduced with, for example, just thefirst and second pressure/flow controls 40, 50, as shown in theembodiment of the system 2000 shown in FIG. 2B. Preferably, thepressure/flow control system 200 includes at least a first pressure/flowcontrol 40 and a second pressure/flow control 50.

Conditions throughout the flow loop, including within the specimen unit10 and/or those experienced by the specimen 12 itself, may be controlledand monitored by a control system 70. The control system 70 may includea processor (not shown in detail) which substantially continuouslytransmits parameters to be monitored and data to be gathered from atleast one, and preferably a plurality of sensors provided at variouspositions within the flow loop. FIG. 2A shows an exemplary placement ofsensors, in which a sensor 72 is provided proximate, and preferablywithin, the specimen unit 10, a sensor 74 is provided upstream of thespecimen unit 10, between the ingress 10 a to the specimen unit 10 andthe first pressure/flow control 40, and a sensor 76 is provideddownstream of the specimen unit 10, between the egress 10 b of thespecimen unit 10 and the second pressure/flow control 50.

The plurality of sensors may serve a variety of functions. For example,the sensor 72 may be a sensor which monitors a condition of the specimen12, such as, for example, a size/diameter, growth rate or wall thicknessof the specimen 12, or a condition of the fluid/media surrounding thespecimen 12, such as, for example, concentration of components offluid/media, or a diffusion of fluid/media (water flux) or of solutes(i.e. fluorescent labeled LDL or dextran, components of the fluid/media)through an outer wall of the specimen 12. Other functions for a sopositioned sensor may also be appropriate. Likewise, the sensors 74, 76may be, for example, sensors which measure a pressure and/or flow rateat a corresponding position in the flow loop. Other types of sensorsand/or sensor placement may also be appropriate. Data collected by theplurality of sensors may be used by the control system 70 to adjustoperation/control parameters for the steady flow system 30, theflow/pressure control system 200, and the like. Arrangement of anynumber and type of sensors may be varied as appropriate based on thecontrol requirements and data gathering needs dictated by a particulartrial.

Numerous types of sensors and actuators may be used to gather the datarequired by the control unit 70. For example, wireless nanotechnology,microelectromechanical systems (MEMS), or electrochemical based systemsmay be integrated at various points within the system 1000 to detect andtransmit data such as, for example, real time metabolite and proteinspresent, % absorption and absorption rates, pressure, flow, and othersuch parameters. This type of technology may also be used as a vehicleto deliver a fluid, cells, or chemicals, such as a drug, to aspecifically targeted area of the specimen 12, to transmit images from aspecific area, or to take other types of readings from a specific areaof the specimen 12 as required. Ultrasound technology may be used tomonitor flow rates, dissipation/diffusion rates, growth rates, and thelike. Strain gauges may be used to monitor pressure/pressurefluctuations throughout the flow loop. The numerous sensors andactuators can be placed in numerous locations throughout the system1000, including both the overall system flow loop and the external flowloop (including the chamber 11).

Other appropriate sensing systems may include, but are not limited to,laser detection systems, and optical detection systems such as, forexample, fluorometers, luminometers, or microscopes. These systems couldalso include probes to measure cell and/or layer integrity on thespecimen 12, and/or to apply electrical stimuli to the specimen 12. Forexample, electrical stimuli may be applied directly to the specimen 12at various locations such as, for example, at a mounting point tomeasure cell layer integrity or enhance growth, function or the like.Various other numbers, types and relative positioning of sensors mayalso be appropriate, depending on the particular conditions to bereproduced, and the amount and type of parameters to be monitored andthe data to be gathered.

The first, second and third pressure/flow controls 40, 50, 60 may takemany forms. For example, as shown in FIG. 2A, the first and secondpressure/flow controls 40, 50 may be, for example, pumps connected tothe flow loop upstream and downstream of the specimen unit 10, and thethird pressure/flow control 60 may be an external pressure/flow controlsystem connected to the specimen unit 10 to exert an external pressureon the specimen 12, or to control a flow of fluid in the chamber 11,such as, for example, the radial flow of fluid through the outercircumferential walls (transmural flow and transport) of the specimen12. In this example, respective drive units (not shown) of the first,second and thirds pressure/flow controls 40, 50, 60 are preferablyindependently controlled. In certain embodiments, the pumps 40, 50 maybe piston-type pumps, such as, for example, bellows pumps, which can beindependently varied in oscillatory motion with typical waveformparameters such as magnitude and phase to produce a desired overalleffect in the specimen unit 10.

Preferably, any oscillatory waveforms or signals can be programmed intosuch pumps which may be used in the first and second pressure/flowcontrols 40, 50. These oscillatory waveforms or signals may include, butare not limited to, for example, a blood pressure waveform, a blood flowwaveform, a diameter waveform, a sinusoidal waveform, a saw-toothwaveform, a square waveform, a frequency control, a slew rate, a dutycycle, a period, a percent systolic or diastolic, harmonic frequencies,magnitude, phase, and the like. Other parameters may also be appropriatefor programming into these exemplary pumps which may be used in thefirst and second pressure/flow controls 40, 50 depending on the effectdesired in the specimen unit 10.

This control of magnitude and phase, amongst other features mentionedabove, in the pertinent parameters provides simulation of a wide rangeof precise and controlled hemodynamic parameters such as WSS, CS,pressure, and the SPA, including in the range in which the most diseasedprone coronary arteries fall (SPA>−250 deg). In other embodiments, thefirst and second pressure controls 40, 50 may include valves, andpreferably occluder valves, which are controlled by the control unit 70to control the flow there through in order to produce similar effects.Since the flow which runs through the flow loop, and, consequently,through the specimen 12, is related to wall shear stress (WSS), and thepressure exerted on the specimen 12 is related to the circumferentialstrain (CS), the pulsatile WSS and the pulsatile CS may be independentlycontrolled and thus may be uncoupled within a certain range.

In alternative embodiments in which the pressure/flow control system 200includes a third pressure/flow control 60, the third pressure/flowcontrol 60 may provide for numerous different, additional conditions tobe reproduced in the specimen unit 10, and thus may take numerousdifferent forms. For example, in certain embodiments, the thirdpressure/flow control 60 may be an external pressure/flow control usedin combination with a chamber 11 surrounding one or more specimen 12.This may include an external flow loop 59 which runs partially throughthe chamber 11, as shown in the embodiment of the system 3000 shown inFIG. 2C, or may include a pump 65, such as the piston or bellows typepumps discussed above with respect to the first and second pressurecontrols 40, 50, used to apply an external pressure to the specimen 12within the chamber 11, as shown in the embodiment of the system 4000shown in FIG. 2D. Alternatively, the third pressure/flow control 60 maybe a combination of an external pump 65 and an external flow loop 59, asshown in the embodiment of the system 1000 shown in FIG. 2A.

This external flow loop 59 may facilitate the introduction and/orextraction of media from the chamber 11, or may be used to induce flowand/or circulation in a particular direction within the chamber 11, suchas, for example, radially, such that the specimen 12 experiencesconditions such as, for example, expansion in a radial direction,bending or other longitudinal deformation, or accelerated or decelerateddiffusion of media through the specimen 12 wall, or facilitate thegeneration of other conditions within the chamber 11 as appropriate. Asshown in FIG. 2A and in more detail in FIG. 4A, the external flow loop59 and external pump 65 may be combined to form the third pressure/flowcontrol 60.

An exemplary external pressure/flow control is shown in more detail inFIG. 4A. In this embodiment, the third, or external pressure/flowcontrol 60 includes an external flow loop 59 coupled to the chamber 11to, for example, induce a circulatory, oscillatory, or pulsatile flow orpressure in the chamber 11, and/or to introduce additional media into orextract media from the chamber 11. Tills exemplary third, externalpressure/flow control 60 may include an external steady flow unit 62 toinitiate and maintain flow through the external flow loop 59. The flowthrough the external flow loop 59 may be a simple recirculation of fluidin the chamber 11. Alternatively, the external flow loop 59 may includeits own reservoir 64 to hold, for example, media to be introduced intothe chamber 11. The external flow loop 59 may also include a varyingflow unit 66 to generate variations in the flow introduced into thechamber 11, such as, for example, a concentrated flow in a particularportion of the chamber 11, or a pulsatile flow to further simulateactual dynamic conditions. The varying flow unit may be a single pistonor bellows type pump, or may be pairs of pumps which operate similar tothe first and second pressure/flow controls 50, 60 described above. Thistype of external flow loop 59 may be combined with a separate externalpiston or bellows type pump 65 which may be separately coupled to thechamber 11, or, alternatively, which may be incorporated into theexternal flow loop 59, to introduce additional forces as discussed abovewith respect to the first and second pressure/flow controls 40, 50.

Numerous different algorithms and methodologies may be applied incontrolling the first, second and/or third pressure/flow controls 40,50, 60 to produce a desired condition in the specimen unit 10 and/orthroughout the flow loop. For example, assuming, simply for purposes ofdiscussion that the steady flow system 30 is a steady flow pump 30 a,and the first and second pressure controls 40, 50 each includepiston/bellows type pumps, as in the systems shown in FIGS. 2A-2E, andthe third pressure/flow control 60 includes an external pump 65, controlof the various pumps may be coordinated to produce a desired condition.If the pumps maintain a mechanical connection through, for example, anadjustable cam that was able to control the timing or phase between theexternal pump and the downstream pump, the external pump would operateat a certain magnitude, as would the downstream pump, but they may peakat different times. Likewise, the pumps may be coordinatedelectromechanically to control there respective timing and phase orsynchrony. Thus, pressure/flow, whether it be upstream, downstream, orexternal, may be controlled by the coordinated action of the pumps attheir respective location(s).

The specimen 12 is preferably positioned within the specimen unit 10using a mounting system 80. The mounting system 80 may be used toappropriately position the specimen 12, whether a chamber 11 is used ornot. Any number/type of mounting systems may be appropriate, dependingon the parameters and characteristics to be reproduced in the specimenunit 10, the properties of the specimen 12 to be studied duringoperation of the flow loop, and the number of specimen 12 to bepositioned in the specimen unit 10. It is also useful and important tobe able to reproduce physical forces) such as, for example, axialstrain, torsion and bending forces, which may be present in an actualphysiological environment on the specimen 12 in the specimen unit 10,whether the specimen 12 is contained in a chamber 11 of the specimenunit 10, or is simply coupled to the flow loop through the mountingsystem 80.

As shown in FIGS. 7A-7D, in certain embodiments, fixed ends of thespecimen 12, which may be, for example, a silicone tube, an expandedPTFE (ePFTE) tube, artery, vein, tissue engineered artery, and the like,may be attached to a rigid tube 14 that can rotate about itslongitudinal axis. The tube 14 and/or specimen 12 are preferably sizedso as to correspond to the actual vessel which it is intended tosimulate. For example, in certain embodiments, the tube 14 and/orspecimen 12 is preferably between approximately 0.5 mm and 30 mm indiameter and various lengths ranging from 1 cm and 80 cm (typically usedin cardiovascular surgery). In other embodiments, the specimen 12 may bea 2D substrate such as a glass slide or other 2D silicone membranestructured appropriate to that which it is to simulate. Again, thechamber 11 may or not be present.

As shown, for example, in FIG. 7A, in certain embodiments the tube 14may be attached to a mount 16 that is coupled to a carriage 18, allowingthe mount 16 to translate in the longitudinal direction. The embodimentshown in FIG. 7A includes a carriage 18 at each end of the specimen unit10, and either one or both carriages 18 may move at a particular time.However, only one carriage 18 may be necessary, depending on conditionsrequired in the specimen unit 10.

A coupler 15 may be attached to both the tube 14 and the mount 16 toprovide for independent movement within a predetermined range of motion.The coupler 15 may then be attached to a drive system 17 such as, forexample, a linear actuator that imposes oscillatory or sinusoidalmotion, a stepper motor, an electrodynamic transducer, and the like, toprovide for motion in accordance with the prescribed conditions to bereproduced in/by the specimen unit 10. More particularly, as shown, forexample, in FIG. 7B, gears 11 and racks 13 may be used to provide linearor torsional motion of the tube 14 and/or specimen 12, with the racks 13directing motion to a corresponding gear 11 to turn the mount 16, towhich the tube 14 and/or specimen 12 is attached, about its longitudinalaxis. A gear 11 may also be used to move a rack 13 to translate themount 16 in an axial direction.

Preferably, the tube 14 has two ends, an upstream and a downstream end,and either or both ends may experience controlled axial strain and/ortorsion. Alternatively, one end may remain fixed and experience nomotion, while the other end experiences some prescribed motion. Morespecifically, the carriage(s) 18 may translate so as to draw the twoopposite ends of the tube 14 and/or specimen 12 apart to induce an axialforce, such as, for example, a component of axial stretching or strain.The carriage(s) 18 may also translate so as to draw the two oppositeends of the tube 14 and/or specimen 12 towards each other so as toinduce a force such as, for example, compressor or bending in the tube14 and/or specimen 12. A mounting of the tube 14 and/or specimen 12 inthe specimen unit 10 using the mounting system 80 is shown in FIG. 7C.

The oscillatory axial strain can be reproduced either with both fixedends of the tube 14 and/or specimen 12 oscillating, or with one fixedend constant and the other end oscillating. The mean axial strain orfixed end(s) of the rube 14 and/or specimen 12 may also be adjusted.That is, variation in axial strain can remain constant, while the meanaxial strain or fixed end position is slowly increased. Torsion may beachieved with both fixed ends of the tube 14 and/or specimen 12rotating, or with one of the two fined ends held constant. In oneembodiment, the tube 14 to which specimen 12 may be attached may beconnected to a gear 13, that provides torsion driven by a rack gear 11.Although the rotation angle can proceed to 360 degrees, the rotationangle is preferably limited to avoid buckling. Preferably, the rotationangle is limited to 0 degrees±45 degrees with both fixed ends rotatingin opposition, or to 0 degrees±90 degrees with one fixed end held in aconstant position. A relationship between axial strain and torsion canbe simulated and varied independent of each other.

Although the exemplary mounting system 80 shown in FIGS. 7A-7C relies onthe drive system 17 described above to provide the movement necessary togenerate axial strain, bending, and/or torsion, other mechanisms whichallow for control of the time varying position of the ends of the tube14 and/or specimen 12 may also be appropriate. Likewise, although onlyone tube 14 and/or specimen 12 is shown mounted using the mountingsystem 80 shown in FIGS. 7A-7C, it would be well understood that themounting system 80 could be readily adapted to receive multiple tubes 14and/or specimen 12, as shown in FIG. 7D. In addition to the longitudinalstrain and torsion in or about the X axis, as described above, incertain embodiments the mount positions may move in 3 dimensions, Y andZ) so as to rotate about the respective axes, Y and Z.

This mounting system 80, which in some embodiments may be considered ahemodynamic axial strain and torsion simulator, may be incorporated intoa flow loop as described herein to reproduce additional hemodynamicforces not reproduced by the various pumps and pressure/flow controls ofthe system so as to provide a more complete physiological hemodynamicenvironment. An embodiment of the system 5000 incorporating thishemodynamic axial strain and torsion simulator shown in FIG. 2E.

In certain embodiments, the mounting system 80 can include additionalcomponents such as additional drive systems 17 coupled to either or bothends of tube 14 to provide longitudinal strain (e.g., stretch) andtorsion (e.g., twist) along the Y axis. Alternatively, such componentsmay be directly or indirectly coupled to the specimen 12 or tubularstructure 1112 to controllably provide Y axis longitudinal stretchand/or twist.

In additional embodiments, the mounting system 80 can include additionalcomponents such as additional drive systems 17 coupled to either or bothends of tube 14 to provide longitudinal strain and/or torsion along theZ axis. Alternatively, such components may be directly or indirectlycoupled to the specimen 12 or tubular structure 1112 to controllablyprovide Z axis longitudinal stretch and/or twist.

Accordingly, embodiments of the specimen holder 10 or pressure flow loopsubsystem 1105, for example using the mounting system 80 or componentsdirectly or indirectly coupled to specimen 12, can provide stretch andtwist in single or opposite directions along individual or combinationsof the X, Y and Z axis of specimen 12 or tubular structure 1112.Embodiments according to the invention can locate such strain and twistalong the X, Y and Z axis at positions intermediate to ends of tubularstructure 1112 (e.g., region A), at branches of specimen 12 (e.g., FIGS.5C, 5D and XI) or for multiple specimens 12 coupled in series orparallel in specimen holder 10.

In certain embodiments, the specimen unit 10 may further includeadditional components to further modify the flow therein when theabove-described components cannot achieve the desired result on theirown. Such additional components may include, for example, jets orinternal fins which could effect helical or secondary flow within thechamber 11 as necessary, or be positioned in the flow loop such that asthe fluid enters the chamber 11, the fluid flow is substantiallyhelical.

Alternative methods or components can be used to generate substantiallyhelical flow, circular flow or wave reflections in specimen 12 ortubular structure 1112. In certain embodiments, systems 1, 5000, 1101can be mounted on mechanical systems that rotate (e.g., horizontally) ata fixed distance around a center point combined with vertical movementrelative to the center point. Such controlled circular and verticalmotion (e.g., merry-go-round) of the systems 1, 5000, 1101 cancontrollably generate a helical flow of fluid in conduit 3701, specimen12 or tubular structure 1112. Further, in certain embodiments, therotation around and vertical movement relative to the center point canbe at a steady or time varying speed (e.g., constant speed, increasingspeed, pulsed speed, sinusoidal speed or the like). Additional movementof the systems 1, 5000, 1101 can be provided by varying the distance ofthe systems from the center point in a controlled fashion. Thus,additional embodiments can selectively provide one or more of theseindividual or reciprocal movements (e.g., tangential, vertical, radialor in combinations thereof) of the system 1, 5000, 1101 around a centerpoint to generate controlled fluid dynamics (e.g., dynamic conditions)according to the viscosities of the fluids and tubular structurestherein.

In certain embodiments, a coupling system 300 may be used to couple thespecimen unit 10 to the flow loop. Although the coupling system 300 isnot required in order for the system 1 to operate as described herein,the coupling system 300 may, for example, allow for quick disconnect ofthe specimen unit 10, and may be adapted to accommodate a specimen unit10 which includes a chamber 11 with either single or multiple specimen12, or may accommodate a specimen unit 10 including a single or multiplespecimen 12 without a chamber 11. The coupling system 300 may alsofacilitate the removal and replacement of specimen unit(s) 10 whilemaintaining necessary sterility of the remainder of the flow loop. Thecoupling system 300 may also allow for quick removal for post-processingof the specimen(s) 12 for further analysis and the like.

An exemplary coupling system 300 is shown in FIGS. 8A-8E. The couplingsystem 300 includes a first coupler 310 which may be separably coupledto a second coupler 320 to form a coupling unit 330. Preferably, thecoupling system 300 includes a coupling unit 330 (i.e., set of first andsecond couplers, 310, 320) positioned on opposite ends of the specimenunit 10 such that the specimen unit 10 may be removed from the flow loopby separating each second couplet 320 from its corresponding firstcoupler 310. In such an embodiment, the first coupler 310 remainsconnected to a portion of the flow loop, while the second coupler 320remains coupled to a portion of the specimen unit 10.

In certain embodiments, the first and second couplers 310, 320 mayinclude corresponding inter-engaging protrusions (male) and recesses(female) which couple the first and second couplers 310, 320 by, forexample, snap fit, or other such means which would facilitate easyengagement and disengagement while maintaining seal and sterilityintegrity. When the corresponding inter-engaging protrusions andrecesses are engaged, their respective through holes are aligned so asto allow fluid to pass therethrough. Upon disengagement of the first andsecond couplers 310, 320, flow inhibitors, such as, for example, simpledisc valves (now shown) inhibit the flow of fluid therethrough, therebymaintaining seal and sterility integrity when separated as well.

It is well understood that any such position and number of correspondingprotrusions and recesses would be appropriate, depending on a number ofspecimen 12 to be sampled and other such considerations. Likewise,although the exemplary first and second couplers shown in FIGS. 8A-8Eare rectangular in shape, it is well understood that a shape of thefirst and second couplers 310 and 320 and the positioning and number ofthe associated protrusions and recesses may be adapted to suit the needsof a particular application. Thus, in this exemplary coupling system300, the fluid/media in the flow loop may be supplied to the couplingsystem 300, and particularly, to the coupling unit 330 positionedupstream of the specimen unit 10, and split so as to supply fluid/mediato twelve specimen 12. Likewise, if multiple specimen units 10 each withits own coupling unit 330 at its ingress 10 a and egress 10 b arealigned with the flow loop, one and/or all of the specimen unit(s) 10may be removed and replaced without compromising critical features suchas, for example, system integrity or sterility.

As discussed above, it is preferable that a coupling unit 330 bepositioned on each end of the specimen unit 10. The first coupler 310includes a number of protrusions 312 extending from a first side 311towards its respective end of the flow loop. In the coupling unit 330positioned upstream of the specimen unit 10, these protrusions 312 arecoupled to flow loop supply lines which receive fluid/media from thereservoir 20. In the coupling unit 330 positioned downstream of thespecimen unit 10, these protrusions 312 are coupled to drain linesentering from the downstream side of the specimen unit 10. The secondside 313 of the first coupler 310 includes a corresponding number ofrecesses 314 which engage corresponding protrusions 322 formed on afirst side 321 of the second coupler 320. The protrusions 322 are fitinto the recesses 314, and an o-ring 319 may be used to improve ascaling characteristic therebetween. The second side 323 of the secondcoupler 320, which preferably faces the specimen unit 10, includes anumber of corresponding protrusions 324 which extend toward the specimenunit 10 and specimen(s) 12 positioned therein so as to supplyfluid/media thereto or drain fluid/media therefrom.

Thus, fluid/media from the flow loop passes through the first and thenthe second coupler 310, 320 of the upstream coupling unit 330, and thenpasses through the specimen unit 10, where the specimen(s) 12 areexposed to the fluid/media. The fluid/media is drained out of thespecimen unit 10 and passes into the second coupler 320 and then firstcoupler 310 of the coupling unit 330 positioned on the downstream end ofthe specimen unit 10, where it is introduced back into the flow loop. Inalternative embodiments, the second side 323 of the second coupler 320may be used when individual chambers 11 and/or specimen(s) 12 are to bedisengaged from the flow loop while others are to remain connected, suchas, for example, during time series analysis, where different chamber(s)11 and/or specimen(s) 12 must be disengaged at different points in timeduring a trial to provide sample data for progression type analysis.

Although the coupling units 330 are shown at upstream and downstreamends of the specimen unit 10, the quick disconnect/reconnect qualitiesand commensurate preservation of sterility afforded by these types ofcoupling units 330 may also be useful at numerous other locationsthroughout the flow loop. For example, a set of coupling units 330 maybe positioned on opposite ends of the first pressure/flow control 40 orthe second pressure/flow control 50 so as to make these systems modularand easily removable/replaceable as well.

The various components of the systems 1, 1101 described above may bejoined to form the flow loop using, for example, tubing. This tubinggenerally comprises any suitable type of laboratory tubing which iscapable of being sterilized, including silicone tubing, or othercomparable laboratory or medical-surgical tubing. The distances betweenthe various components and the corresponding length of the tubing may bechosen so as to minimize the total volume of fluid used. Preferably,these lengths are calculated to provide a maximum flow rate, and toavoid turbulence in the system, based upon boundary layer theory, asknown to those skilled in the art. Generally, it is preferable tominimize the amount of fluid used in order to reduce the costs of mediautilization, drug treatment, and cell by-product (such as, but notlimited to, proteins, metabolites and like) detection and the like.

Systems for reproducing a hemodynamic environment in accordance withother embodiments of the invention as broadly described herein will nowbe discussed with respect to FIGS. 3A-3D. The systems and combinationsof components discussed above with respect to the embodiments of thesystem shown in FIGS. 2A-2E are readily adapted to the embodiments shownin FIGS. 3A-3D. Thus, for example, the coupling system 300, mountingsystem 80, and control system 70 as described above may each be appliedto the systems as shown in FIGS. 3A-3D. Thus, there are any number ofpossible combinations of these components, as well as their placementwithin embodiments of the system, and, simply for ease of discussion,any duplicative description is omitted.

The system 6000 shown in FIG. 3A includes a specimen unit 10 with aspecimen 12 mounted therein by a mounting system 80 and coupled to aflow loop by coupling units 330. A reservoir 20, first pressure/flowcontrol 40 and second pressure/flow control 50 cause fluid/media to flowfrom the reservoir 20 through the flow loop as described above. However,steady flow from the reservoir 20 through the first and secondpressure/flow controls 40, 50 is now provided by a steady flow system 30comprising a pair of upstream and downstream pressure/flow controloccluders 35-38 provided upstream and downstream of the specimen unit 10which provide for steady flow of fluid/media into the flow loop andappropriate flow into and out of the first and second pressure/flowcontrols 40, 50

These pressure/flow control occluders 35-38, which may be, for example,pinch valves, or flow occluders and the like, positioned upstream anddownstream of the specimen unit 10 occlude flow and pressure in acontrolled oscillatory manner, thus allowing for steady or mean flowwithout a steady flow pump.

In operation, when one occluder per pressure/flow control 40 or 50 isopen, the other is preferably closed. Thus, for example, when the firstupstream occluder 35 is open, the second upstream occluder 37 is closedand pump 40 can eject or push fluid toward the open occluder 35 which isconnected to the specimen 12 at an appropriate pulsatile or other suchrate as dictated by a required condition. Likewise, to fill or supplythe pump 40 occluder 37 is open while occluder 35 is closed, allowingpump 40 to draw fluid from the reservoir 20 through the open occluder37, where it may be held by the pump 40 and closed occluder 37 until,for example, sufficient fluid has been collected therein to operate thepump 40 to create the particular flow dictated by the desired condition.The downstream occluders 36, 38 operate in a similar manner. This allowsfor control of various hemodynamic parameters such as flow, pressure,and diameter and consequent hemodynamic forces in the specimen unit 10.

Alternatively, a variety of conditions may be achieved while maintaininga mean pressure by controlling the first pressure/flow control 40 andupstream occluders 35, 37 along with the second pressure/flow control 50and downstream occluders 36, 38 to essentially maintain a mean pressurewhile still permitting control of flow and pressure. One exemplarymanner in which this may be achieved is by closing upstream occluder 37and opening upstream occluder 35. This will allow fluid to move towardthe specimen unit 10 and pressure and flow will continue to increase inthe specimen unit 10 until downstream occluder 36 is opened and 38 isclosed (or open), thus allowing fluid to exit the specimen unit 10 andreducing pressure accordingly. As pressure and flow reach the desiredvalue, upstream occluder 35 may be closed, and upstream occluder 37 maybe opened. This allows a mean pressure and flow to be maintained in thespecimen unit 10 through appropriate, coordinated timing of the openingand closing of the occluders 35-38.

A system for reproducing a hemodynamic environment in accordance withanother embodiment of the invention as broadly described herein is shownin FIG. 3B. The system 7000 shown in FIG. 3B is similar to the system6000 shown in FIG. 3A. However, the system 7000 includes a thirdpressure/flow control 60 which includes an external flow loop 59separately coupled to the specimen unit 10 as described above. However,the external flow loop 59 shown in FIG. 3B obtains steady flow in theexternal flow loop 59 from a pair of external pressure/flow controloccluders 68, 69 (rather than an external steady flow pump). Likewise,the systems 8000 and 9000 shown in FIGS. 3C and 3D are similar to thesystem 7000 shown in FIG. 3B. However, the system 8000 includes anexternal pressure control 65 as discussed above, in combination with anexternal flow loop 59 which now includes another pair of externalpressure/flow control occluders 61 and 63. This additional pair ofexternal pressure/flow control occluders 61, 63 may be employed tofurther maintain constant pressure or flow in the chamber 11 if sodesired. In the system 9000 shown in FIG. 3D, the third pressure/flowcontrol 60 is simply an external pump 65 externally coupled to thechamber 11.

As can be well understood, the various means set forth herein may becombined as necessary and expedient to achieve a desired result. Thus,for example, steady flow may be provided both in the flow loop and inthe external flow loop by a number of different component(s) and/orcombination(s) of components, such as, for example, a steady flow pump,or a pairing of pressure/flow occluders and their operation with acorresponding pressure/flow control or pump. Likewise, a third, externalpressure control may or may not be included in the pressure/flow controlsystem, and may include, for example, simply an externally appliedpressure/flow control in the form or a pump, or a partial or fullexternal flow loop, or a combination thereof. The coupling system andmounting system discussed above may be applied to any of thecombinations of components as appropriate/required to provide enhancedutility and/or ease of use.

Likewise, any of these systems may include a variety of other componentsnot shown in detail in these particular figures, such as, for example, aflow damper, or noise filter, that reduces vibrations or noise in thefluid flow. Resistors, such as flow restrictors or clamps that restrictor reduce flow, may be used to increase pressure in the specimen unit 10or other location within the flow loop if the resistor is appropriatelypositioned, such as downstream of the downstream pump if this conditionis desired in the specimen unit 10. Capacitors, such a chamber that hasair and fluid in it and acts as a compliance chamber, can be placedupstream or downstream of the specimen unit 10, preferably downstream,to help adjust various hemodynamic parameters such as the impedancebetween flow and pressure.

The various system components, such as, for example, tubing,reservoir(s), and pumps, may be made of a variety of materials. Incertain embodiments, these components may be made from disposablematerials such as, for example, plastic, polypropylene, PETG, and thelike to facilitate providing and maintaining a sterile environment, aswell as ease of set up and change out of test trials. In otherembodiments, these components may be made of non-disposable materials,such as, for example, metals, to provided enhanced durability,structural integrity, and the like. In still other embodiments, thesecomponents may be made of a combination of disposable and non-disposablematerials, that can be sterilized by, for example, ETO, autoclave, gammairradiation, and the like, such materials preferably being non-toxicmaterials.

An exemplary operation of the systems shown in FIGS. 2A-2E and 3A-3Dwill now be discussed with reference to FIGS. 9A-9C. As shown in FIG.9A, first, the specimen unit 10 is coupled to the flow loop (S100),preferably using the coupling units 330 as described above. The steadyflow system 30 is activated to draw fluid/media from the reservoir 20,which is holding fluid and/or media therein, into the flow loop (S200),and the pressure/flow control system 200 is also activated (S300) sothat as the fluid/media is drawn through the upstream coupling unit 330and into the specimen unit 10, the appropriate dynamic conditions arepresent in the specimen unit 10. Alternatively, the pressure/flowcontrol system 200 may be activated first, followed by the steady flowsystem 30, or the two systems may be activated simultaneously, dependingon the requirements of a particular trial. The introduction of thefluid/media into the specimen unit 10, and particularly thecharacteristics of the fluid/media associated with pressure and/or flow,as well as the conditions within the specimen unit 10, and particularlythose associated with pressure and/or flow of the fluid/media in thespecimen unit 10, are established by the pressure/flow control system200 based on parameters preset in the control unit 70. As the specimen12 experiences the dynamic conditions reproduced in the specimen unit10, the sensors collect data and transmit the data to the control system70 for monitoring and analysis (S400). The control unit 70 maydynamically monitor, control, and adjust the operation of the steadyflow system 30 and the pressure/flow control system 200 as necessarybased on its substantially continuous analysis of the data collected.

The fluid/media then passes out of the specimen unit 10, again, at apressure and/or flow condition established by the pressure/flow controlsystem 200 based on control parameters in the control system 70. Theoutgoing fluid/media passes through the downstream coupling unit 330 andback towards the sampling port 21 of the reservoir 20. At the samplingport 21, the fluid/media is directed to either the reservoir 20, anoutflow portion 20 b of the reservoir 20, or a holding tank outside theflow loop, again based on preset parameters stored in the control system70 and characteristics measured by the sensor 23.

The system 1000 continues to operate in accordance with the controlparameters set by/in the control system 70 until a preset condition orparameter is reached (S500). The governing parameter or condition, whichmay be preset in the control system 70, may be, for example,time/elapsed time, cycles, a remaining level of fluid and/or media inthe reservoir 20, a concentration or other characteristic of thefluid/media as it is returned to the sampling port 21 of the reservoir20, and other such parameters and/or conditions. When the presetcondition has been satisfied, the steady flow system 30 and thepressure/flow control system 200 are deactivated (S600, S700), thecontrol system 70 collects and analyzes any remaining data as required(S800), the specimen unit 10 is decoupled from the flow loop (S900) andpost-processing analysis is performed. When other conditions areincluded, such as, for example, axial stretch and/or torsion componentsprovided by the mounting system 80, these auxiliary systems may beactivated as necessary after the flow conditions are set. The sensorscan initiate sensing as required to either provide feedback or nofeedback to the control system 70 throughout operation of embodiments ofthe system as required.

As discussed above, the processor 70 may be used to control the variouscomponents of embodiments of the system to produce a desired conditionor set of conditions in the specimen unit 10 and/or at various locationsthroughout the flow loop. The control system 70 may control embodimentsof the system to operate in numerous modes, including, for example, afirst mode in which the control system 70 controls embodiments of thesystem based on manually entered or preset parameters/algorithms, withlittle to no feedback from various sensors which may be positionedthroughout the flow loop, and no commensurate dynamic adjustment (anopen loop control mode). The control system 70 may also controlembodiments of the system in a second mode in which the manually enteredor preset parameters/algorithms may be dynamically adjusted based onfeedback received from the numerous sensors positioned throughout theflow loop (a closed loop control mode). Feedback may include, forexample, pressure, flow, diameter, strain, metabolite production, andother such measurements related to a particular condition/set ofconditions. Numerous other parameters may also be monitored and fed backto the control system 70 to provide for the dynamic adjustment of thecontrol parameters and algorithms applied by the controller based on theparameters dictated by a particular condition/set of conditions. Othercontrol modes, including a combination of the open and closed loopcontrol modes, may also be appropriate. These control modes arediscussed in more detail below.

FIG. 9B is a flow chart of the operation of the controller throughoutthe process shown in FIG. 9A, in accordance with an embodiment of theinvention. It is assumed that at least one, and preferably a pluralityof dynamic conditions and associated control parameters/algorithmsproducing the consequent hemodynamic forces are previously stored in amemory portion (not shown) of the control system 70 for selection by anoperator at the initiation of a particular trial. In alternativeembodiments, conditions and/or control parameters may be selected orentered manually. Such manually entered conditions/parameters mayinclude, for example, flow magnitude, pressure, magnitude, phaserelation) and other such parameters which may produce a desiredhemodynamic condition.

As shown in FIG. 9B, first a hemodynamic condition/set of conditions isselected (S10). The control parameters/algorithms associated with aselected condition/set of conditions may be retrieved from a previouslystored set of control parameters/algorithms (S30), or may be manuallyentered (S25), based on requirements dictated by a particular trial andother such considerations (S20). For example, a specific hemodynamicregion in which certain flow and pressure conditions will have certainassociated wall shear stresses and circumferential strain levels may bechosen to produce a patient specific condition, such as, for example, adistressed coronary artery with a typical large phase difference betweenpressure and flow, or a healthy condition in which a phase differencebetween flow and pressure is relatively small. As discussed above, theseconditions and associated control parameters may be previously stored inthe control system 70. Likewise, parameters such as flow magnitude,pressure magnitude, phase relation, and the like may be manuallyentered, and then resulting conditions calculated by the control system70, if desired.

Once the control parameters/algorithms have either been retrieved frommemory (S30) or manually entered (S25), the control system 70 sends thecorresponding control parameters/algorithms to the various affectedcomponents (S40) such as, for example, the steady flow system 30, thefirst second and third pressure/flow controls 40, 50, 60 and theircorresponding components which are included in the pressure/flow controlsystem 200, the mounting system 80 to provide for appropriate axialstrain and/or torsion, and any other components linked to the flow loopwhich should be controlled in a given manner to produce the selectedhemodynamic condition/set of conditions. These control parameters mayinclude, for example, output voltages or currents with appropriateoscillatory patterns (such as, for example, sinusoids or blood pressurewaveforms) to produce the desired conditions.

As the control system 70 operates embodiments of the system, itdetermines whether or not feedback has been received (S50). If nofeedback has been received from the sensors, the control system 70checks to see if any new/additional control parameters have beenmanually entered (S55). If new control parameters have been entered(S25), the new control parameters are received by the control system 70and transmitted to the components (S40). If new control parameters havenot been entered, the control system 70 can determine if the trial iscomplete (S70), and, if not, continues to transmit the valid controlparameters to system components (S40). This process continues until thecontrol system 70 determines that the trial is complete (S70).

If feedback is received from the sensors (S50), the control system 70determines if adjustment to the control parameters is required based onthe feedback (S60). To accomplish this, the control system 70 may, forexample, conduct a comparison of the control parameters as originallyestablished to a set of measured parameters. Alternatively, the controlsystem 70 may receive the various feedback parameters, and perform acalculation to determine actual dynamic conditions at a particularlocation compared to conditions which were initially established forthat location. If, based on these comparisons/calculations, the controlsystem 70 determines that no adjustment is required, the control system70 then determines whether the trial is complete (S70), and, if not,continues to transmit the valid control parameters to the systemcomponents (S40). If feedback is received from the sensors (S50) andadjustment of the control parameters is required based on thecomparisons/calculations, then the control parameters are adjusted (S65)and the adjusted control parameters are transmitted to the systemcomponents (S40). This process continues until the control system 70determines that the trial is complete (S70).

As set forth above, the various embodiments of the system describedherein may be adapted to receive numerous different types of specimenand be operated and configured in a variety of different manners basedon the requirements dictated by a particular trial. For illustrativepurposes, operation of the system 2000 shown in FIG. 2B, in which acompliant specimen including, for example, a compliant silicone tubelined with endothelial cells so as to be representative of an actualvessel, in-vivo, with similar mechanical properties such as, forexample, modulus of elasticity, compliance, and the like, has beenmounted in the specimen unit 10 for drug screening and testing will nowbe discussed in more detail. It is well understood that this is just oneexample of the many applications of each of the various systems setforth herein, and is not meant to in any way be construed as so limitingthe application or operation of embodiments of the system as embodiedand broadly described herein.

If, for example, the silicone tube lined with endothelial cellsdiscussed above is to be subjected to a particular hemodynamic conditionfor testing, appropriate parameters are set to produce such a condition.In this example, a healthy hemodynamic condition may be represented by aWSS of 10±10 dynes/cm² at a pressure of 70±20 mmHg and a circumferentialstrain represented by a change in diameter of ±4%, yielding an SPA of 0degrees at a frequency of 1 Hz. As discussed above, these controlparameters may be manually entered, or they may be stored in a memoryportion of the control system 70 in association with a given hemodynamiccondition, and accessed as necessary prior to the initiation of a trial.

Once the appropriate hemodynamic condition is selected and thecorresponding control parameters are made available, the control system70 controls to the steady flow pump 30 a to operate to initiate acirculation of fluid through the flow loop. The first and secondpressure/flow controls 40, 50, which, in this example, are likely to bebellows pumps, oscillate to produce oscillatory waveforms correspondingto the required dynamic conditions. This may be accomplished by) forexample, the first pressure/flow control 40, considered in this exampleto be the upstream pump, creating an increase in flow and pressuredirected toward the specimen unit 10, while the second pressure/flowcontrol 50, considered in this example to be the downstream pump,simultaneously creating an increase in flow and pressure directed towardthe specimen unit 10. The coordinated action of the upstream anddownstream pumps and the resultant pressure and flow conditions producedin the specimen unit 10 result in an oscillatory component at or abovethe 0 degree SPA associated with a healthy hemodynamic condition for thesuch a specimen.

The oscillatory waveforms generated by the coordinated action of theupstream and downstream pumps in this example may be varied by varyingthe action of the upstream and downstream pumps accordingly. Thus, forexample, rather than directing an increase in pressure and/or flowtoward the specimen unit 10, one of both of the upstream and downstreampumps may instead operate to draw fluid collected in the specimen unit10 out of/away from the specimen unit, thereby producing adifferentiated effect on the specimen mounted therein. In thisparticular example, if bellows pumps are employed at the upstream anddownstream positions, this may be accomplished by allowing the bellowsportion of the pumps to fill with fluid from the flow loop through theaction of the steady flow pump 30 a which maintains a mean flow throughthe flow loop concurrent with the action of the upstream and downstreampumps, and then controlling a release of fluid from the bellows towardthe specimen unit as required to produce the desired effect. Or,alternatively, the bellows may be filled from the specimen unit 10 sideof the respective pump and the release of the collected fluid into theflow loop controlled to produce an alternately directed effect.

As described above, in this particular example, the steady flow pump 30a maintains a mean flow throughout the flow loop, concurrent with theaction of the upstream and downstream pumps. Thus, as the upstream anddownstream pumps collect and discharge fluid toward/away from thespecimen unit 10, at least some, if not all of the fluid running throughthe pumps as they operate is replenished with circulating fluid. Asfluid leaves the downstream pump, it travels toward the reservoir 20,where, in this particular example, a portion thereof is periodicallysiphoned off at the sampling port 21 for sampling. The remainder of thefluid is then returned to the reservoir 20 for recirculation in thisparticular example, although, as discussed above, in other applications,this return fluid may be fully or partially diverted to an outflowportion 20 b or holding tank rather than recirculated. Thisrecirculation of fluid and operation of the various pumps as describedabove is continued in accordance with the established algorithms until apreset stop condition is achieved. In an example such as this, in whicha specimen is undergoing drug testing, this stop parameter is often timebased, i.e., exposure of the specimen 12 to a particular set ofconditions for a given amount of time, based on actual interaction ofsuch drugs in-vivo. However, as discussed above, this stop condition mayvary based on requirements dictated by a particular trial.

This is just one example of how one of the embodiments of the inventionmay be employed for a drug screening and testing trial on a compliantsilicone tube lined with endothelial cells. It is well understood thatthe various other components described herein may also be applied toembodiments of the system to augment the capability of that system andprovide further variation in the dynamic conditions to which a specimenmay be exposed. For example, addition of a third pressure/flow control60, which may include a pressure/flow control pump, a full external flowloop, or a combination thereof, may provide for further variation of theflow environment created within the specimen unit 10 and commensurateadditional combinations of hemodynamic force. The addition of torsionand/or axial strain through implementation of the capabilities of themounting system 80 may further expand the sets of condition(s) which maybe created in the specimen unit 10 and experienced by a particularspecimen. Numerous different environments and parameters may bemonitored and/or control algorithms adjusted based on a number, type andplacement of a variety of sensors throughout the selected system and thecapabilities of the control system 70.

FIGS. 10A-10H provide graphical representations of the various stress(WSS) and strain (CS) conditions which may be achieved by the varioussystems FIGS. 2A-2E and 3A-3C, graphically depicted in terms of pressure(P), diameter (D) and flow rate (Q). More specifically, FIGS. 10A-10Hdemonstrate control of magnitude, phase, and frequency of flow,pressure, and diameter waveforms in a specimen 12 such as, for examplean artificial or silicone artery, and the unique conditions that may beachieved by the system 100 in the chamber 11. The various conditions andcombinations of conditions graphically depicted in FIGS. 10A-10H aretabulated in Table 1 below.

TABLE 1 Various conditions shown in FIGS. 10A-10H, where an oscillatorycondition is shown as T, and a constant condition is shown as F. Q P D Aand B T T T C T F F D T F T E T T F F F T T G F T F H F F T (not shown)F F F

In Table 1, an oscillator condition for one of the parameters Q, P or Dis shown as True or “T” state, while a constant or non-time varyingcondition is shown as a False or “F” state. For example, a condition inwhich there is oscillatory flow Q (True state) with no change inpressure P or diameter D (False state) as shown in line C of Table 1 andgraphically depicted in corresponding FIG. 10C may now be achieved dueto the capabilities provided by the combination of components providedin the systems shown in FIGS. 2A-2E and 3A-3C. Further, a condition inwhich there is oscillatory flow Q (True state), oscillatory diameter D(True state), and no change in pressure P (False state) as shown in lineD of Table 1 and graphically depicted in corresponding FIG. 10D may nowbe achieved due to the capabilities provided by the combination ofcomponents provided by the systems shown in FIGS. 2A-2E and 3A-3C.

FIG. 11 shows a side view of a specimen, shown as a tubular structure1112, in a specimen unit (not shown) in accordance with an embodiment ofthe invention. Specimen 1112 is represented as a tubular structurehaving a length L′. Specimen 12, described above, includes, but is notlimited to, a tubular structure 1112. As used herein, tubular structure1112 includes any three dimensional structure capable of passing fluidfrom one location to another. This includes shapes of any section foundin the cardiovascular system in humans or animals or any shapes ofsections, including but not limited to C, I, T, Y of FIGS. 5A-5D and 11.Tubular structures 1112 further include any shapes of sections found inhumans or animals that serve to transfer or pass fluid from one locationto another. For example, tubular structures can include, but are notlimited to, aortas, arteries, arterioles, capillaries, venules, veins,vena cavas, pulmonary arteries and pulmonary veins. Tubular structurescan further be synthetic, partially porous, permeable, grooved,microgrooved, hybrid biological/synthetic and/or electrospun.

Region A as shown in FIG. 11 represents a portion or subsection ofspecimen or tubular structure 1112. Specimen 1112 has a diameter ofapproximately D(t) over a length L which is ≦L′. In accordance with oneembodiment of the invention, a sample has pressure P and flow Q, if themeasured pressure P and flow Q are substantially within ΔP and ΔQ of thevalues of P and Q over the Region A. Hence, region A represents aportion of tubular structure 1112 in which pressure is substantiallybetween P±ΔP/2, flow is Q±ΔQ/2, and diameter is D±ΔD/2, and a specimenis said to have dynamic conditions P, Q and D, if the measured values ofP, Q and D over a region A are substantially within the ratiosΔP/P_(Range), ΔQ/Q_(Range) and ΔD/D_(Range), respectively, whereP_(Range), Q_(Range) and D_(Range) can be, for example, mean values ofthe potential ranges of pressure, flow and diameter for specimen 1112.In preferred embodiments, ΔP/P_(Range)≦0.35, and preferablyΔP/P_(Range)≦0.25, and more preferably ΔP/P_(Range)≦0.15 and even morepreferably ΔP/P_(Range)≦0.05, similarly ΔQ/Q_(Range)≦0.35, andpreferably ΔQ/Q_(Range)≦0.25, and more preferably ΔQ/Q_(Range)≦0.15 andeven more preferably ΔQ/Q_(Range)≦0.25, and similarly ΔD/D_(Range)≦0.35,and preferably ΔD/D_(Range)≦0.25, and more preferably ΔD/D_(Range)≦0.15and even more preferably ΔD/D_(Range)≦0.05.

FIG. 12 shows examples of cross-sections of tubular structures 1112according to various embodiments of the invention. The cross-sections oftubular structures can be circular, ovular or elliptical, even lobeshaped (such as a figure eight). Other embodiments of the invention mayinclude, tubular structures having a nearly two dimensional flattenedribbon shape with an ovular and/or rippled shaped cross-section as shownin FIG. 12.

In a preferred embodiment of the invention, specimen 1112 is notcompletely rigid in that the shape of its cross-section may vary inresponse to sufficiently large variations in dynamic conditions such aspressure P(t), flow Q(t) will structures WSS, circumferential strain(CS), stretch or Length (L), twist/torque (T) and so forth. Hence,tubular structures preferably have at least some flexibility in thesense that the diameter D(t) (as generally defined herein) can vary inresponse to sufficiently large variations in pressure P(t), flow Q(t),stretch or Length (L), and/or twist/torque (T) along a selecteddirection of measurement.

FIGS. 12 and 13A demonstrate how diameter D(t) as used herein can be aparameter generally indicative of the shape of a cross-section oftubular structures. The shape of the tubular cross-section may benon-circular, such as elliptical or ovular, in which case the diameterD(t) represents a parameter indicative of variations in thatcross-sectional shape. For example, parameter D(t) can represent theinner diameter, the outer diameter, the tubular structure's wallthickness along one or more directions of the cross-sectional area alonga selected direction as shown in FIG. 12. The selected direction ofmeasurement can be in any direction with respect to the cross-sectionalarea.

FIG. 13A shows several examples of how a direction of measurement can beselected for the measurement of the parameter D(t) as well as how themeasurement of the inner and/or outer diameter of a cross-section oftubular structures can be accomplished according to alternativeembodiments of the invention. For example, FIG. 13A shows parameters D₁and D′₁ representing the inner and outer diameter, respectively, of across-sectional area of a tubular structure as measured along thedirection 1. Similarly, parameters D₂ and D′₂ represent the inner andouter diameter, respectively, of the tubular structure as measured alongthe direction 2. The parameter D(t) can be combinations of D₁, D′₁, D₂,and/or D′₂. For example, parameter D(t) might be the thickness of thewalls of the tubular structure along direction 1, namely, D₁(t)-D′₁(t).Also, diameters can be measured along additional directions and thosevalues combined by controller 70 and/or independently monitored bycontroller 70 as independent feedback signals.

The tubular structure may also be a multi-layer structure, in which caseparameter D_(xy) can represent the inner diameter of layer x alongdirection y and D′_(xy) can represent the outer diameter of layer xalong direction y.

FIG. 13B is a schematic cross sectional view of a human blood vessel(e.g., artery or vein), which is an example of a multi-layer tubularstructure as broadly defined herein, which shows how the parameter D(t)inner and/or outer diameters of a cross-section of multi-layer tubularstructures can be accomplished according to alternative embodiments ofthe invention. Arteries and veins follow substantially the samehistological makeup. The inner most layer is an inner lining called theendothelium, followed by a second layer of subendothelial connectivetissue. This is followed by a third layer of vascular smooth muscle,which is highly developed in arteries. Finally, there is a fourth layerof connective tissue called the adventitia, which contains nerves thatsupply the muscular layer, as well as nutrient capillaries in the largerblood vessels.

Parameters D₁₁ and D′₁₁ represent the inner and outer diameters,respectively, of a cross-sectional area of a first layer of amulti-layer tubular structure as measured along the direction 1.Similarly, parameters D₂₁ and D′₂₁ represent the inner and outerdiameters, respectively, of a second layer of the multi-layer tubularstructure as measured along the direction 1.

Parameters D₁₂ and D′₁₂ represent the inner and outer diameters,respectively, of a cross-sectional area of the first layer of themulti-layer tubular structure as measured along the direction 2.Similarly, parameters D₂₂ and D′₂₂ represent the inner and outerdiameters, respectively, of the second layer of the multi-layer tubularstructure as measured along the direction 2.

Tubular structures are further categorized into those which respond todynamic conditions in a substantially consistent manner and those thatdo not.

Referring to FIG. 14, dynamic conditions g_(i)(t) can be measured atvarious locations on systems, in accordance with embodiments of theinvention. Feedback FB_(i)(t) can include signals indicative of dynamicconditions at locations other than regions A of specimen 1112. Suchdynamic conditions g_(i)(t) might, from time to time, be referred to assystem or global dynamic conditions.

As used herein, stable dynamically responsive tubular structures aretubular structures whose dynamic conditions at region A aresubstantially repeatable for a given set of global dynamic conditionsand/or local dynamic conditions for a system 1101 according toembodiments of the invention.

A system can be trained using a first tubular structure with a stableand dynamic responsivity. If the relationship between the physicalstructure of the first and any subsequent tubular structures is knownand these subsequent tubular structures have a stable dynamicresponsivity, then global dynamic conditions can be translated bycontroller 1103 to yield dynamic conditions at the subsequent tubularstructures a priori. For example, if a system 1101 is trained using afirst tubular structure, and global dynamic conditions (e.g., FB_(i)(t))and input information has been linked (in accordance with, for example,FIG. 29), then a second stable tubular structure with an outer wallthickness twice that of the first tubular structure, but the same innerdiameter of the first tubular structure could be inserted in specimenunit 10 of pressure/flow loop subsystem 1105. Controller 1170 can thenperform the appropriate translations to yield local dynamic conditionsat a corresponding region A of the second tubular structure, providedthe responsivity of a second tubular structure with twice the wallthickness is known a priori. Translation of properties between stabletubular structures can be ascertained as known to those skilled in theart, for example, fluid dynamics and fluid mechanics.

As discussed above, sensors, detectors, transmitters, receivers and/ortransceivers (referred to herein from time to time as “sensors”) can bearranged within, on and/or around pressure/flow loop subsystem 1105 tosense, detect and/or measure various dynamic conditions at variouslocations in pressure/flow loop subsystem 1105 and/or to transmitinformation. The locations of such sensors will yield feedback signalsFB_(i)(t) corresponding to types of dynamic conditions (examples ofwhich are shown in FIGS. 17A and 17B) that could be considered globaldynamic conditions of system 1101. As with a single region A, knownvariations of stable tubular structures can be linked to global dynamicconditions by in controller 1103 in accordance with embodiments of theinvention.

If a given controller 1103 is trained using a stable tubular structurehaving an architecture, for example, C, I, T, Y or some otherarchitecture, then controller 1103 may include processing which mapsthose global dynamic conditions to multiple or alternative regions A fora given tubular structure in accordance with embodiments of theinvention. For example, using a stable tubular structure, multipleregions A1-A5 can be selected can be selected during a training processand dynamic conditions at their respective locations can be linked toglobal dynamic conditions measured at the input ( G _(jn)) and theoutput ( Gout) located at the input and output of specimen holder 10,respectively, as shown in FIG. 15.

These system sensors include sensors, transmitters, receivers,detectors, transceivers, etc., and can sense, detect, measure, transmitand/or receive information which can be directly or indirectlyassociated with dynamic conditions at any location. System sensors canbe as small or smaller than nanosensors or be large or moresophisticated systems, such as an MRI, PET or other systems, as will bediscussed herein.

FIG. 16 shows an alternative block diagram of a system 1101 with aspecimen or tubular structure 12, such as system 1 of FIG. 1A accordingto another embodiment of the invention. System 1101 includes acontroller 1103 and a pressure/flow loop subsystem 1105. Controller 1103receives input data or information corresponding to desired dynamicconditions and translates that information to a set of N control signalsf_(j)(t), j=1, 2, 3, . . . , N. The set of N control signals f_(j)(t)can be a single control signal or multiple control signals.Pressure/flow loop subsystem 1105 includes pressure/flow loop componentssuch as the various elements, devices or subsystems in the embodimentsdiscussed herein. Pressure/flow loop components can include, forexample, pressure/flow control system 200 (FIG. 1A) and any elements,devices or subsystems contained therein as well as other elements,devices or subsystems contained in the embodiments of the systemsdiscussed herein, such as steady flow systems, specimen units, sensors,reservoirs, pumps, upstream or downstream pumps, steady flow pumps,occluders, external pressure controllers, axial strain system, slidercarriage, torsion systems and any other elements in the pressure/flowcontrol systems. Control signals f_(j)(t) in turn are input topressure/flow loop subsystem 1105 where each control signal f_(j)(t)controls and/or adjusts one or more of the pressure/flow loopcomponents.

As discussed above, the set of control signals f_(i)(t) can be a singlecontrol signal or multiple control signals for driving the variouscomponents of the pressure/flow loop subsystem 1105. The variouspressure/flow loop components of the pressure/flow loop subsystem 1105can be controlled with respective control signals f_(j)(t). In oneembodiment, controller 1103 outputs a separate control signal f_(j)(t)for each component to be controlled in the pressure/flow loop subsystem1105.

Alternatively, some or all of the components that make up thepressure/flow loop subsystem 1105 could be mechanically coupled, suchthat an adjustment to one component using a control signal f_(j)(t) willcause a predetermined adjustment in another component via such amechanical coupling. This allows for the adjustment of multiplecomponents using fewer control signals f_(j)(t) than the number ofcomponents in the pressure/flow loop subsystem 1105. Such mechanicalcouplers are described in related U.S. Pat. No. 7,063,942 filed on Oct.9, 2001, and incorporated by reference in its entirety.

In addition, the individual components in the pressure/flow loopsubsystem 1105 can also be non-mechanically coupled and adapted tocommunicate with each other independently of controller 1103, including,for example, feedback and status information of one or more of theindividual components or feedback information. Such coupling ofinformation or data among individual elements or components of thepressure/flow loop subsystem 1105 can include pressure/flow loopcomponent feedback information, such as the status of respectivepressure/flow loop components (see, for example, F_(fb) in FIG. 27)and/or feedback data or information, such as FB_(j) or otherinformation. Such non-mechanical coupling provides a non-mechanicalimplementation of mechanical coupling, including but not limited to themechanical coupling described in U.S. Pat. No. 7,063,942. Localprocessing can also be used, as discussed, for example, with respect toFIGS. 28 and 27.

Controller 1103 includes, for example, any control systems discussedherein including, for example, control systems 70 in FIGS. 1A-3D and6A-6D. Controller 1103 can receive input information or input datacorresponding to desired dynamic conditions such as desired pressure,flow and diameter, desired SPA's, sample dimensions and structuralinformation related to a sample or samples. Controller 1103 can alsoreceive feedback information such as feedback signals FB_(j)(t)corresponding to one or more measured dynamic conditions such aspressure, flow, diameter, velocity, presence, amounts and concentrationsof particles, nano-particles, organic and inorganic molecules and/or anybiological substances, drugs or materials introduced into the fluid inpressure/flow loop subsystem 1105 or grown or emerging from the specimen12 and/or the growth of biological materials in specimen unit 10 or asotherwise discussed herein.

Input information can also include information regarding the pathologyand degree of pathology to be simulated, the structure and properties ofthe sample or samples, the length of time a sample should be subjectedto a particular set of dynamic conditions, the rate and manner in whichthe dynamic conditions change or progress over time, the composition ofthe fluid and the rate and manner in which the composition of the fluidchanges over time. Controller 1103 can also serve to couple varioustypes of dynamic conditions such as pressure (P), flow (Q), diameter(D), length or stretch (L) and twist/torque (T) to shear stress (WSS),circulation strain (CS), and in turn the SPA, and vice versa asdiscussed herein in accordance with preferred embodiments of theinvention.

Input information can also include information corresponding tocharacteristics of signals representing the dynamic conditionsincluding, for example, the frequency, phase, amplitude, slew ratesand/or duty cycle of the dynamic conditions, which controller 1103translates into control signals f_(j)(t) which in turn drive de variouscomponents of the pressure/flow loop subsystem 1105 in accordance withembodiments of the invention. The dynamic conditions may becharacterized by discrete or continuous random variables or stochasticvariables.

Feedback signals FB_(j)(t) are received by controller 1103, whichcorrespond to one or more measured dynamic conditions in pressure/flowloop subsystem 1105, as discussed herein with respect to variousembodiments of the invention. Feedback signals FB_(j)(t) can be dynamicconditions actually measured at region A of specimen 1112 (as shown inFIG. 11, in accordance with an embodiment of the invention. Feedbacksignal FB_(j)(t) can be measured dynamic conditions at other locationsin pressure/flow loop system 1105, either upstream or downstream fromspecimen 12 in pressure/flow loop system 1105. Controller 1103 receivesfeedback signals FB_(j)(t) and in turn can produces control signalsf_(j)(t) for pressure/flow loop subsystem 1105.

FIGS. 17A and 17B show examples of various forms or types of dynamicconditions g(t). Forms or types of dynamic conditions refers to adirectly or indirectly measurable time varying physical condition of orrelated to tubular structures and/or fluids passed therethrough broadlydefined herein. Examples of various forms of types of dynamic conditionsg(t) which can be produced by system 1101 include pressure P(t), flowQ(t) wall shear stress WSS(t), circumferential strain CS(t,) diameterD(t), length or stretch (L) and twist/torque (T) as broadly definedherein in accordance with the embodiments of the invention. System 1101can simulate one, two, three or more forms or types of dynamicconditions in states that may occur in biological as well asnon-biological systems.

FIG. 17B lists types that are linked to dynamics of fluid materials.These include, but are not limited to, for example, concentration offluid material (C_(fm)), expression of fluid material (E_(fm)), amountsof fluid material (A_(fm)), velocity of fluid material (V_(fm)) and flowof fluid material (Q_(fm)).

As above, region A represents a portion of the specimen 12 or tubularstructure 1112 is said to have dynamic conditions g₁, g₂, . . . g_(n) ifthe measured values of g₁, g₂, . . . g_(n) over a region A aresubstantially within the ratios of

$\frac{\Delta \; g_{1}}{g_{1}{Range}},{\frac{\Delta \; g_{2}}{g_{2}{Range}}\mspace{14mu} \ldots \mspace{14mu} \frac{\Delta \; g_{n}}{g_{n}{Range}}},$

respectively, where g₁Range, g₂Range . . . g_(n)Range can be, forexample, mean values of the potential ranges of g₁, g₂, . . . g_(n),respectively. In preferred embodiments, over a region A,

${\frac{\Delta \; g_{j}}{\Delta \; g_{j}{Range}} \leq {.35}},$

and preferably

${\frac{\Delta \; g_{j}}{\Delta \; g_{j}{Range}} \leq {.25}},$

and more preferably

$\frac{\Delta \; g_{j}}{\Delta \; g_{j}{Range}} \leq {.15}$

and even more preferably

$\frac{\Delta \; g_{j}}{\Delta \; g_{j}{Range}} \leq {{.05}.}$

FIG. 18 shows examples of classes of dynamic conditions that can besimulated by systems according to various embodiments of the invention.Classes of dynamic conditions refer to the location of the tubularstructure at which a set of dynamic conditions to be simulated mightoccur. Dynamic conditions that occur in vivo are referred to herein fromtime to time as in vivo dynamic conditions. In vivo dynamic conditionsinclude dynamic in vivo bio conditions and hemodynamic conditions.Dynamic in vivo bio conditions may include, for example, dynamicconditions that cells, tissues, or organs, experience in vivo other thanhemodynamic conditions. Dynamic conditions can also includenon-biological dynamic conditions found in tubular structures as broadlydefined herein in accordance with alternative embodiments of theinvention.

FIG. 19 shows a block diagram of controller 1103 which includes atranslator 1113 and a dynamic parameter or dynamic condition generator1117. Input information can include dynamic conditions represented byg_(j)(t) according to an embodiment of the invention. For example,dynamic conditions g₁(t), g₂(t) and g₃(t) could be pressure P(t), flowQ(t), and diameter D(t), at a region A, respectively. Input informationcan be information which is used to characterize the dynamic conditionsg_(j)(t). Input information can be used to retrieve certain preselecteddynamic conditions g_(j)(t) stored in controller 70 and/or generatedynamic conditions and/or associate or link dynamic conditions or statesof physiology as required to produce control signals for pressure/flowloop subsystem 1105 in accordance with embodiments of the invention.

FIG. 20 shows a translator 1113 which receives dynamic conditionsg_(j)(t) and translates those dynamic conditions to N control signalsf_(t)(t) . . . f_(N)(t). The number and characteristics of the controlsignals f_(j)(t) depend on the architecture implemented forpressure/flow loop subsystem 1105 as will be discussed in accordancewith various embodiments of the invention.

FIG. 21 shows physiological coronary flow Q(t) and pressure P(t) to beproduced by system 1101 at, for example, specimen 12 of FIG. 18. In thisexample, the state includes types of dynamic conditions, pressure P(t),flow Q(t) and diameter D(t) where diameter represents the outer diameterof a tubular structure. The class of dynamic conditions is in vivohemodynamic coronary conditions. A representative signal correspondingto pressure P(t) can be generated digitally with signal processingtechniques or actually measured by sampling over a period T or othermethods as known to one of ordinary skill in the art. Controller 1103can perform a Fast Fourier Transform (FFT) on P(t) to yield theamplitude and phase of P(t) for the first and higher order harmonics. Inembodiments of the invention, amplitude and phase of at least the firstharmonic is determined and/or utilized, and preferably the first twoharmonics, and more preferably the first three harmonics, and morepreferably at least the first 4-10 or more harmonics are determined andutilized.

Controller 1103's capability to vary one dynamic variable while keepingothers constant, for example, to vary pressure P(t) while maintainingflow Q(t) and/or diameter D(t) constant, enables controller 70 to “dialup” a preselected set of dynamic variables.

FIG. 22 shows an exemplary pressure/flow loop subsystem 1105 for system1101 of FIG. 1B in accordance with an embodiment of the invention.Pressure flow loop subsystem 1105 includes bellows pumps 405 a and 405 bpositioned at the upstream and downstream ends 10 a and 10 b,respectively of the specimen unit 10 in concert with occluder valves35-38 respectively positioned upstream and downstream of each of thebellows pumps 405 a, 405 b to generate an exemplary dynamic condition Aset of control signals f₁-f₄ which correspond to the desired conditionare generated by the control system 70 to control the occluder valves35-38, and dynamic control signals f₅ and f₆ control operation of eachof the bellows pumps 405 a, 405 b to generate the desired condition inthe specimen unit 10. For ease of discussion, the valves 35-38 areeither fully open or fully closed. However, it is well understood thatthe values 35-38 may at any given time be partially open/closed, andthat appropriate slew rates may be applied to the opening/closing of anyof the valves 35-38 to generate different conditions in the specimenunit 10 as required.

FIGS. 23 a-23 d show various stages of bellows pumps 405 a and 405 b andFIG. 24 shows states during one cycle, or period T of operation has beendivided into four segments 0-T/4, T/4-T/2, T/2-3T/4, and 3T/4-T. Oneperiod of operation can correspond to cycle of or heart beat or asdescribed herein in accordance with embodiments of the invention. Attime T=0, as shown in FIG. 23 a, the upstream pump 405 a is fullyexpanded and full of fluid, and the downstream pump 405 b is fullycontracted, thus having little to no fluid capacity. Both of theupstream valves 35 and 36 are closed, while the downstream valves 37 and38 are open, thus containing the fluid between the valve 35, through thefirst pump 405 a and the specimen unit, and to the valve 36. As thesystem moves to the condition T/4, with the valves 37 and 38 open, theupstream pump 405 a contracts to push the fluid into the specimen unit10, and the downstream pump 405 b expands to prepare to draw fluid awayfrom the specimen unit 10 and into the pump 405 b once the valve 36 isopened. Thus, at time T/4, the valves 35 and 36 are open, the valves 37and 38 are closed, the upstream pump 405 a is contracted, and thedownstream pump 405 b is expanded.

As the system moves from this arrangement/condition at T/4, as shown inFIG. 23 b, towards T/2, the valves 35 and 36 open, the valves 37 and 38close, the upstream pump 405 a expands once again fill with fluid, andthe downstream pump 405 b contracts to expel fluid from the pump 405 band out into the downstream end of the flow loop towards the reservoir20. As the system moves from this arrangement/condition at T/2, as shownin FIG. 23 c, towards 3T/4, the valves 35 and 36 once again close, thevalves 37 and 38 once again open, the upstream pump 405 a contracts topush fluid into the specimen unit 10, and the downstream pump 405 bexpands to draw fluid from the specimen unit 10 and into the pump 405 b.

From this point, one cycle, or “pulse,” is completed as the system movesto from 3T/4, as shown in FIG. 23 d, to time T, where the valves 35 and36 open, the valves 37 and 38 close, the upstream pump 405 a expandsonce again fill with fluid, and the downstream pump 405 b contracts toexpel fluid from the pump 405 b and out into the downstream end of theflow loop towards the reservoir 20.

It is noted that, in this particular example, the flow of fluid throughthe specimen unit 10 is a substantially regular pulsatile flow in whichfluid is drawn into the specimen unit 10, held there for a given (small)amount to time, and then drawn out into the flow loop. In thisparticular example, simply for ease of discussion, the expansion andcontraction of the bellows pumps 405 a, 405 b is shown to occursubstantially about the centers of the bellows. However, by expandingand/or contracting the pumps 405 a, 405 b in different directions fromthose shown in FIGS. 23 a-23 d, such as by forcing all of the fluid heldin the bellows to flow in a single direction which may be opposite thatof the fluid held in the other bellows, and/or by varying therate/timing of the opening and closing of the valves 35-38, numerousdifferent conditions may be generated. More specifically, as the fluidflows into and out of the specimen unit 10 through the interaction ofthe fluid pushed into and drawn out of the specimen unit 10 by theupstream and downstream pumps 405 a, 405 b, numerous differentcombinations of pressure and/or flow rate may be generated as the fluidis forced to occupy the same space and/or change direction as it“collides” in the specimen unit 10, or is simultaneously drawn out ofthe specimen unit 10 a from both the upstream and downstream ends 10 a,10 b.

An exemplary dynamic condition in which pressure and flow aresubstantially in phase, in which SPA is essentially 0°, is shown in FIG.25A. In this essentially healthy condition, the valve 35 would initiallybe closed and the pump 405 a full of fluid which is pumped through openvalve 37 into the specimen unit 10, out of the specimen unit 10 troughopen valve 36, where it is stopped by closed valve 38, pushed back intothe specimen unit 10 through the action of the pump 405 b, and thendrawn out again through open valves 37 and 38 into the flow loop andtowards the reservoir 20.

Another exemplary dynamic condition in which pressure and flow are 90°out of phase, or an SPA of essentially 90° representative of a somewhatdiseased condition, is shown in FIG. 25B. Still another exemplarycondition in which pressure and flow are 180° out of phase, or an SPA ofessentially 180° representative of a more severely diseased condition,is shown in FIG. 25C. These conditions may be generated by varying thedirection(s) in which the fluid is moved by the pumps 405 a, 405 b intoand out of the specimen unit 10, and the varying degrees of pressureand/or flow disturbance or acceleration experienced as a result.

FIG. 26 shows a schematic diagram of features of a bellows pump 400 suchas bellows pumps 405 a and 405 b of FIG. 22. Pump 400 is one example ofa pump that can be implemented in systems in accordance with alternativeembodiments of the invention. A first end 406 a of bellow 405 is fixed,for example, to a first support 410. The first support 410 is shown inFIG. 26 as attached to a structure 415 that renders it substantiallyunmovable. The second end 406 b of the bellow 405 is attached to amovable support 420.

The movable support 420 is attached to a movable plate 425, which is inturn movable by means of a drive system 430 comprising a linear motor431 and magnetic plate 435 in accordance with an embodiment of theinvention. The linear motor 431 interacts with the magnetic plate 435 tomove the movable plate 425 and therewith the movable support 420 and thesecond end 406 b of the bellow 405. Other types of drive systems mayalso be appropriate.

The bellow 405 may be made, for example, of plastic, such aspolypropylene, or silicon.

The drive system 430, and in particular, the linear motor 431 can bedriven by one or more control signals. An encoder unit 440 may bearranged to include an encoder 440 a attached to the movable plate 425,and a reader 440 b, which senses a position of the movable plate 425 andprovides the feedback signal f_(fb) to the pump controller 2701. Theencoder unit 440 may be, for example, a mechanical encoder, an opticalencoder, a capacitive encoder, a magnetic encoder or a laser encoder,which would include a laser and corresponding reader.

In this exemplary pump, blood flows into the bellows pump 400 in adirection of arrow 36 in FIG. 26 via orifice 445 and exits the pump 400in a direction of arrow A2 via orifice 450. The pump 400 is providedwith the control signal, such as control signal f₅(t) discussed above,received from controller 70, which controls the pumping of the pump 400to provide the desired flow characteristics. That is, the drive system430, including linear motor 431 and magnetic plate 435, move the movablesupport 420 and the second end 405 b of the bellows pump 405 to createthe desired pumping effect in response to the control signal f₅. Thefeedback signal f_(fb) indicative of the position of the movable plate425 is provided by the encoder unit 440 to pump controller 2701 toensure the desired pumping effect is being created.

The drive system 430 is driven by a control signal, such as controlsignal f₅(t) discussed above, received from controller 70. The controlsignal f₅(t) controls the current to the linear motor 431 via pumpcontroller 2701 shown in FIG. 27, according to an embodiment of theinvention. Pump controller 2701 includes motor controller 2703 andamplifier 2705. Motor controller 2703 may reside in controller 70. Inalternative embodiments of the invention, motor controller 2703 mayreside in dynamic condition generator 1117 and/or translator 1113. Motorcontroller 2703 may independently control one or multiple motors 431.Feedback signal f_(fb) can be received from encoder 440 by pumpcontroller 2701 at motor controller 2703 and/or amplifier 2705. Anexample motor controller 2703 is SPii Plus HP Series motion controllerby ACS Motion Control. Examples of motors 430 includes AC servo/DCbrushless motors, DC brush motors nanomotion piezo-ceramic motors, stepmotors and servo motors. Motor 430 preferably has sub-nanometerresolution such as those used in semiconductor manufacturing, waterinspection, or flat panel display assembly and testing.

FIG. 28 shows controller 70 with processor 2711 coupled to pumpcontroller 2701 and memory 2715 in accordance with an embodiment of theinvention. Motor controller 2703 in pump controller 2701 can be used toprocess input information received by controller 70. See also FIGS. 18,28, 19 and 22. Motor controller 2703 may include a local processor 2709and memory 2707 such as cache memory or other types of memory. Referringto FIGS. 19 and 28, the roles of dynamic condition generator 1117 andtranslator 1113 can be shared to varying degrees by processor 2711 andlocal processor 2709 in motor controller 2703. Pump controller 2701 maytake on the bulk of the processing in controller 70 so that processor2711 functions merely to synchronize generation of dynamic conditionsg_(j)(t) and translate the dynamic conditions to control signalsf_(j)(t) based on input information to controller 70 according to oneembodiment of the invention. In alternative embodiments, processor 2711may perform a greater portion of the processing in controller 70. Forexample, processor 2711 can generate process input information to linkto pump controller 2701 to yield control signals f_(j)(t). The dynamicconditions g_(j)(t) can be linked to input information in controller 70at, for example, local memory 2707 and/or in memory 2715.

Referring again to FIGS. 18, 19 and 20 in accordance with embodiments ofthe invention, control signals f_(j)(t) for pressure flow loop subsystem1105 are determined by operating with a first set of controls signalsf_(j)(t) input to pressure flow loop subsystem 1105 and measuring aresulting first set of dynamic conditions and linking that first set ofcontrol signals with the resulting first set of dynamic conditions. Oneor more of the control signals are then slightly varied to yield asecond set of control signals, measuring a resulting second set ofdynamic conditions and linking the second set of control signals to theresulting second set of dynamic conditions. This process is repeated toform a discrete set of dynamic conditions linked to a corresponding setof control signals which can be stored, for example, as a lookup tablein controller 70. The number of sets of dynamic conditions can varydepending on the desired flexibility of system 1101. A variety ofinterpolation techniques can also be used to interpolate between sets ofdynamic conditions to provide corresponding sets of control signalsthereby yielding a fully flexible “dial-up” system 1101 capable ofproducing sets or states of dynamic conditions between those determinedusing the above approach in accordance with yet another embodiment ofthe invention.

FIG. 29 shows steps that may be implemented to develop sets of controlsignals corresponding to dynamic conditions and/or input informationaccording to an embodiment of the invention. Step S1301 involvesselection of an initial set of control signals, which can be written asa vector F₁ (t) of k control signals, namely, F₁ (t)=(ƒ₁₁(t), ƒ₁₂(t) . .. ƒ_(1k)(t)). As an example, an initial set of control signals can be asinusoidal signal for pumps, as described in reference to variousembodiments of the invention. Occluders in dynamic pressure/flowsubsystem could be arranged to receive control signals as shown, forexample, in FIGS. 22 and 24. At step S1305, the initial set of controlsignals F₁ (t) are input to the pressure/flow loop subsystem 1105.

Step S1306 involves measuring an associated or corresponding set orstate of dynamic conditions, which can be written as a vector G₁ (t) ofM dynamic conditions, namely, G₁ (t)=(g₁₁(t),g₁₂(t) . . . g_(1M)(t)).Step S1307 involves linking or associating the resulting dynamicconditions g₁ _(j) (t) to the initial stage or set of control signals f₁_(j) (t). Linking can include storing a lookup table in memories 2701and/or 2705 (see FIGS. 28 and 27) of controller 70 or 1103 according toan embodiment of the invention. Step S1307 may also include storingcharacteristics of the measured dynamic conditions g₁ _(j) (t)associating pathological as well as the shape or characterization of thesignals representing the dynamic conditions to the control signals. StepS1309 involves adjusting or perturbing one or more control signals f₁_(j) (t) to yield a second set or state of control signals F₂ , whichinclude element control signals f₂ _(j) (t), then measuring theresulting second set or state of dynamic conditions G₂ , which includeelement dynamic conditions g₂ _(i) (t).

This process can be repeated to produce a desired number of linksbetween input information and/or dynamic conditions G_(m) and controlsignals F_(m) in accordance with embodiments of the invention. Forexample, implementation of steps S1301-S1309 involves assigning pumps inpressure/flow loop subsystem 1105, a sinusoidally varying control signalcorresponding to position of the bellows versus time (as discussed withrespect to bellows pumps 400 in FIG. 26) with a frequency approximatelyequal to a base heart rate. Step 1309 may then involve varying the phaseof one of the bellows pumps with respect to the other bellows pump toestablish a next set of control signals F_(m) and measuring theresulting set of dynamic conditions G_(m) .

Alternatively, step 1309 might involve varying the amplitude or strokelength of one of the bellows pumps with respect to the other toestablish a next set of control signals F_(m) . Also, in accordance withlinear and non-linear interpolation techniques, sets or states ofdynamic conditions can be linked to associated sets of control signalsto enable “dial-up” dynamic conditions.

Controller 70, 1103 can further be trained to produce dynamic conditionsthat evolve over time. This includes adjusting the frequency, phase oramplitude of the first and/or higher order harmonics of one or moredynamic conditions over multiple periods T of pulses.

For example, the first order frequency ω₁ of one or more types ofdynamic conditions can vary over time TT^(i) in a predetermined manner.For example, the resting heart rate might be at 70 beats per minute. Thedynamic conditions such as pressure P(t), flow Q(t) and/or diameter D(t)could gradually change from a first order frequency of 70 Hz to 130 Hzover a time span of minutes, hours, etc. The rate and progression ofchange for different order harmonics may differ for any one dynamiccondition as well as for different types of dynamic conditions P(t)versus Q(t), D(t), etc. This holds for the phase θ_(j) of the first orhigher order harmonics of one or more dynamic conditions.

FIG. 30 shows variations over time in the first order harmonic ω₁(t) ofa dynamic variable g(t) which can be produced in accordance with anembodiment of the invention. At t=0, ω₁(t) might correspond to a restingheart rate ω₁(0)=1/T (say 70 cycles per second), where T is the periodof the heartbeat. The first order frequency ω₁(t) then increases toω₁(t1) (say 120 cycles per second) over multiple periods T (e.g. 5T,10T, 100T, 1000T . . . ) at time t₁ (t₁=5T, 10T, 100T, 1000T . . . ).Between time t₁ and t₂, the first order harmonic ω₁(t) remainsrelatively constant at ω₁(t2) then decreases to ω₁(t)=ω₁(t3) (say 100cycles per second) at time t₃. Between time t₃ and t₄, ω₁(t) againremains relatively constant at ω₁(t3) and then increases back to ω₁(t1)at t₅ and continues to increase for t>t₅. The above holds for higherorder frequencies ω_(1j)(t) as well in accordance with embodiments ofthe invention.

FIG. 31A shows an example of the variations in time of the phases θ_(j)of the first three harmonics for j=1, 2 and 3 of a dynamic conditiong(t). The corresponding first three harmonics ω₁(t), ω₂(t) and ω₃(t)could remain constant or themselves varied over time in accordance withalternative embodiments of the invention. The number of harmonics whosephase and amplitude are utilized can be preferably 1, and morepreferably at least 2 and more preferably at least 3 and more preferablyat least 4-10. The same holds for multiple forms or types of dynamicconditions it being understood that the frequency ω₁(t) and phaseθ_(j)(t) of one dynamic condition g₁(t) may differ from the frequencyω₁(t) and phase θ_(j)(t) of a second or additional dynamic variablesg₁(t) in accordance with embodiments of the invention. In theseembodiments of the invention, system 110 can be used to simulate aperson or mammal exercising or exerting effort in any physical activity,experiencing shock, disease or any other situations which couldnaturally occur.

FIG. 32 shows variations of the _(j)th harmonic amplitude G_(j) of adynamic condition g(t) where

$\begin{matrix}{{g(t)} = {\sum\limits_{j = 1}^{N}\; {G_{j}{\mu_{j}\left( {{\omega_{j}t} + \theta_{j}} \right)}}}} & (1)\end{matrix}$

where μ_(j)(ω_(j)t+θ_(j)) are normalized basis functions of dynamiccondition g(t), such as sinusoids. Just as ω_(j)(t) and θ_(j)(t) mayvary over time T, the amplitude G_(j) can vary over time in accordancewith embodiments of the invention.

FIG. 32 presents one example of how the amplitude of the _(j)th harmonicof dynamic condition g(t) (initially G_(j)(0) at t=0), increases toG_(j)(t₁) at t=t₁. The amplitude G_(j)(t) remains at G_(j)(t₁) untilt=t₂, then increases to G_(j)(t₃) at t=t₃, where it remains until t=t₄at which point it decreases to G_(j)(t₁) at t=t₅. Dynamic condition g(t)may be one of the types (FIGS. 17A and 17B) of dynamic conditions fromone or more states or classes (FIG. 18) of dynamic conditions.

FIG. 31B shows an example of how amplitudes of the first three harmonicsof a dynamic condition may vary with time as well as the correspondingdynamic condition in real time. At t=0, the first order amplitudeG₁(0)>G₂(0), the second order amplitude G₂(0) is about 0.8 G₁(0), andthe third order G₃(0) is about half of G₁(0). The presence of the higherorder terms for dynamic variable G(t) are apparent as variations in theplot of G(t) versus time. As t approaches t₁, the second and third orderamplitudes G₂(t) and G₃(t) approach zero, which results in the dynamiccondition G(t) varying more sinusoidally.

Referring to Equation (1) above, G(t) represents a vector of N types ofdynamic variables or conditions (FIGS. 17A and 17B) g_(i)(t), that isi=1 . . . N where N≧2. Hence,

G ≡(g ₁(t),g ₂(t) . . . g _(N)(t))

System 1101 produces an experience G at region A in a tubular structurein accordance with embodiments of the invention. An experience cancorrespond to actual dynamic conditions experienced at region A oftubular structures in vivo or actual dynamic conditions experienced atregion A of tubular structures (including non-biological dynamicconditions) that are not in vivo. In addition, an experience cancorrespond to dynamic conditions which are used to train or condition atubular structure.

For example, three experiences G ^(A)(t), G ^(B)(t) and G ^(C)(t) can berepresented as:

G ^(A)=(g ₁ ^(A)(t),g ₂ ^(A)(t) . . . g _(N) ^(A)(t))

G ^(B)(t)=(g ₁ ^(B)(t),g ₂ ^(B)(t) . . . g _(N) ^(B)(t) . . . g _(N′)^(B)(t))

G ^(C)(t)=(g ₁ ^(C)(t),g ₂ ^(C)(t) . . . g _(N) ^(C)(t) . . . g _(N′)^(C)(t) . . . g _(N″) ^(C)(t))

where the experiences may be actual in vivo, actual non-biological,training or conditioning and/or combinations thereof in accordance withembodiments of the invention. The types of dynamic conditions FIGS. 17Aand 17B) for experiences G ^(A), G ^(B) or G ^(C) are not necessarilythe same. For example, g₁ ^(A) is not necessarily the same as g₁ ^(B)(t)or g₁ ^(C)(t). Also, the number of dynamic conditions N, N′ or N″ can bedifferent in accordance with embodiments of the invention.

FIGS. 33A and 33B show representative frequencies ω_(ij)(t) andamplitudes G_(ij)(t) for three different physiological experiences i=A,B, and C, respectively. Training time corresponds to the length of timethat a set of dynamic conditions are to be produced at a tubularstructure before they are repeated. Total training time TTT^(i)corresponds to the total time a tubular structure is subjected to a setof dynamic conditions for the i^(th) physiological experience.

FIG. 34 shows an example of how systems such as systems 1 and 1101produce a single experience using three dynamic variables P′(t), Q′(t)and D′(t) and which exhibit a pattern of variations over a training timeTT^(i) which is repeated for a total training time TTT^(i)=4 TT^(i).Hence, P′, D′ and Q′ can represent, for example, the amplitude, phase orfrequency of the pressure, flow and diameter at a specimen or tubularstructure 12, 1112. In accordance with embodiments of the invention,total training time TTT^(i) can be multiple training times TT^(i) andcan include fractions of training time TTT^(i). For example,TTT^(i)=xTT^(i), where x is a real number, for example x=0.3, 1 5/3, 20,100, 1000 . . . and so forth.

FIG. 35A shows how systems 1, 1101 can be used to produce dynamicconditions which would be experienced by in vivo tubular structures in apatient with a particular patient history while undergoing aphysiological experience. In particular, the dynamic conditions arereproduced at a specimen or tubular structure 12 or 1112 inserted intosystems such as systems 1, 1101 in accordance with embodiments of theinvention.

Step 3501 involves inputting physical characteristics of an in vivotubular structure located in a patient and inputting patient historyinformation. FIG. 35B shows exemplary patient history information.

Step 3503 involves either selecting a non in vivo tubular correspondingto the in vivo structure, or removing the in vivo tubular structure fromthe patient. Step 3505 involves inserting the selected tubular structureinto system 1, 1101. Step 3507 involves selecting a physiologicalexperience, examples of which are shown in FIG. 35B.

Step 3509 involves implementing the selected physiological experienceusing system 1, 1101 with the selected tubular structure and optionallyadding, subtracting and/or altering the fluid and/or fluid material inthe system 1, 1101 for a time TTT^(i). Step 3511 involves testing,removing and/or outputting resulting fluid from the flow loop of system1, 1101 and/or testing removing and/or outputting the resulting tubularstructure from the system 1, 1101.

In accordance with additional embodiments of the invention, it should beunderstood that the number and types of dynamic variables used duringtraining time TT^(i) for a particular physiological experience can varyduring a training time TT^(i). For example, system 1101 could produce 2dynamic variables g₁(t) and g₂(t) while measuring or monitoring theresulting third dynamic variable g₃(t), then produce dynamic variablesg₂(t) and g₃(t) while measuring or monitoring the resulting firstdynamic variable g₁(t). The measured/monitored dynamic variable canserve as feedback signals FB₁ (see, for example, FIG. 18) for controller1103 in accordance with various embodiments of the invention.

The physiological experiences can be selected to train a tubularstructure (biological or non biological as discussed above) and arerepresented by two or more types of dynamic conditions (or variables)for any state or class of dynamic conditions. For example, physiologicalexperience A might have a training time TT^(A)=24 hours with dynamicconditions G ^(A)(t) made up of a set of the three dynamic conditionspressure P′(t), diameter D′(t) as broadly defined herein, and flow Q′(t)experienced by a tubular structure located in the pulmonary artery of a60-year old athletic male Caucasian (patient history) reported overmultiple training times TT^(A). Physiological experience B might, forexample, correspond to a training time of TT^(B)=1 week with dynamicconditions G ^(B)(t) made up of a set of two dynamic conditionsexperienced by a tubular structure located in the large intestine of a25-year old athletic paraplegic (patient history). Again, other examplesof physiological experiences are listed in FIG. 35B, it being understoodthat system 1101 is not necessarily limited physiological experienceslisted therein.

Controller 1101 can be characterized by its flexibility, the classes(FIG. 18) of dynamic conditions and/or types (FIGS. 17A and 17B) ofdynamic conditions that can be produced at a given region of a tubularstructure.

Controller-Flexibility

For a given pressure/flow loop subsystem 1105, controller 1103 may betrained to provide a single state of dynamic conditions, (a single statecontroller) in accordance with embodiments of the invention. Similarly,for a given same pressure/flow loop subsystem 1105, controller 1103 maybe trained to provide discrete states or sets of dynamic conditions, (adiscrete controller) in accordance with other embodiments of theinvention. Also, for a given same pressure/flow loop subsystem 1105,controller 1103 may be trained to provide multiple discrete andcontinuous states of dynamic conditions (a hybrid controller) inaccordance with embodiments of the invention. Similarly, for a givenpressure/flow loop subsystem 1105, controller 1103 may be trained toprovide a single physiological experience (FIG. 34) (a single experiencecontroller), multiple physiological experiences (a multi-experiencecontroller), and a hybrid of discrete physiological experiences and alsothe flexibility to dialup various states of dynamic conditions, (ahybrid experience controller).

Controller—Type or Form (FIGS. 17A and 17B) of Dynamic Conditions

For a given pressure/flow loop subsystem 1105, controller 1103 can betrained to output control signals f_(j)(t) which yield certain types offorms FIGS. 17A and 17B) of dynamic conditions. For example, controller1103, can be trained to output control signals that produce g₁(t),g₂(t), g₃(t) and g₄(t) at a region A of a tubular structure, where g₁(t)is pressure P(t), g₁(t) is flow Q(t), g₃(t) is the wall thickness alonga first direction and g₄(t) is the circumferential strain at region A.

Controller—Class of (FIG. 18) of Dynamic Conditions

For a given pressure/flow loop subsystem 1105, controller 1103 can betrained to output control signals f_(j)(t) which yield states of aparticular class (FIG. 18) of dynamic conditions. For example, ifcontroller 1103 is trained to output control signals f_(j)(t) to a givenpressure/flow loop subsystem 1105 which yield one or more states in thedynamic bio class of conditions for example, controller 1103 is anon-invivo condition controller, and system 1101 is a dynamic biocondition system.

For a given pressure/flow loop subsystem 1105, system 1101 may bereferred to as a single state system if controller 1103 is a singlestate controller, a discrete state system if controller is a discretestate controller, a hybrid system if controller is a hybrid controller,and a dial-up system if controller is a dial-up controller in accordancewith embodiments of the invention.

Similarly, for a given pressure/flow subsystem 1105, system 1101 may bea single experience system if controller 1103 is a single experiencecontroller, a multi-experience system if controller 1103 is amulti-experience controller, and a hybrid experience system, ifcontroller 1103 is a hybrid experience controller.

The method and systems described herein can be characterized byadditional/alternative means in accordance with embodiments of theinvention as shown in FIGS. 36, 37, 39 and 38. FIGS. 36 and 37 showblock diagrams of system 1101 with controller 1103 and a pressure/flowsubsystem 1105 which together generate a flow loop 2105 of fluid asbroadly defined herein. System 1101 of FIG. 36 includes a specimen unit10 in accordance with embodiments of the invention, whereas system 1101in FIG. 37 shows a block diagram with controller 1103 and apressure/flow subsystem 1105 with a flow loop 2105 of fluid, but withouta specific specimen unit 10. Instead, a region of the flow loop 2105itself serves as the region A of a tubular structure (e.g., FIG. 11) inaccordance with embodiments of the invention. Controller 1103 is trainedto generate control signals f_(j)(t) which when input to pressure flowsubsystem 1105 produce various dynamic conditions and/or physiologicalexperiences at a tubular structure in accordance with embodiments of theinvention.

FIGS. 38 and 39 show system 1401 with pressure/flow loop subsystems 1105in accordance with embodiments of the invention. Here pressure/flow loopsubsystem 1105 does not include flow loop fluid but does include aconduit 3701, which operatively couples pressure/flow loop subsystemcomponents, in accordance with various embodiments of the invention.Conduit 3701 can include any tubes, pipes, cylinders, tubular structuresand any other coupling components described herein with respect tosystems such as systems 1 and 1101 and others known to those of ordinaryskill in the art. As with system 1101 of FIG. 36, system 1101 of FIG. 39includes a specimen unit 10 in accordance with an embodiment of theinvention. Similarly, as with system 1101 of FIG. 37, system 1101 ofFIG. 38 does not include a specimen unit 10. Instead, a region of thepressure/flow loop subsystem 1105 itself serves as the region A of thetubular structure (FIG. 11) in accordance with embodiments of theinvention. Again, controller 1103 is trained to generate control signalsf_(j)(t) which when input to pressure/flow loop subsystems 1105 producevarious dynamic conditions at a region A in pressure/flow loop subsystem1105.

FIG. 40 shows system 1101 with sensors 1, 2 n, A and Bn. Although foursensors are shown as an example, purposes, number and types can begreater or smaller than four. Sensors 1) 2 n, A and/or Bn include, forexample transmitters, receivers, transmitter/receivers, transducers,detectors and other sensors. Fluid sensors as used herein are sensors inthe fluid which are added to the pressure/flow loop subsystem of FIGS.38 and 39 or which comprise the flow loop shown in FIGS. 36 and 37.

System sensors (A) include any transmitters, receivers,transmitters/receivers, transceivers, transducers, detectors, as well asany devices which can be used to detect images or measure any one ormore parameters related directly or indirectly to one or more dynamicconditions, such as those listed in FIGS. 17A and 17B and discussedherein.

FIGS. 41A-44C show three electrode configurations for measuring theconductivity of a fluid and/or a monolayer in a tubular structure 1112,in accordance with embodiments of the invention. In the embodiment ofFIG. 17C, electrodes 1200 and 1202 are placed on opposite sides of thetubular structure 1112 and are connected to a voltage source 1204. Inthe embodiment of FIG. 17D, ring electrodes 1206 and 1208 are spacedapart and extend around at least a portion of the circumference of thetubular structure 1112, and preferably around the entire circumferenceof the tubular structure 1112. In the embodiment of FIG. 17E, electrodes1200 and 1202 are placed on one side of the tubular structure 1112.

The three electrode configurations of FIGS. 41A-44C measure theconductivity of the fluid inside the tubular structure 1112 and/or amonolayer inside the tubular structure 1112 along different directions.For example, the configuration shown in FIG. 17E is particularly usefulfor measuring the conductivity of a monolayer (not shown) grown on theinside surface of the tubular structure 1112. Such a conductivityreading could be used, for example, to measure the functionality and/orthe integrity of the monolayer in the tubular structure 1112.

The voltage source 1204 can be a direct current source or an alternatingcurrent source. Thus, the term “conductivity”, as used herein, includesthe measurement of resistivity, impedance and reactance.

System sensors A can include more complex detecting, measuring and/orimaging systems, including, but not limited to, digital cameras, MRI,NMR, and PET systems, microscopes, ultrasound systems, including 3D or4D ultrasound imaging systems, chemical sensor systems, gas analyzers,electromagnetic detecting/measuring and/or imaging systems and any otherfluid material (e.g., FIG. 17B, detecting/measuring and/or imagingsystems.

System nanosensors (Bn) represent nanosensors, nanotransmitters,nanoreceivers, nanotransceivers, nanotransducers, nanodetectors, as wellas any devices which can be used to detect, image or measure one or moreparameters related directly or indirectly to one or more dynamicconditions, including, but not limited to, those listed in FIGS. 17A and17B.

Referring back to FIG. 40, fluid sensors 1 include any transmitters,receivers, transmitters/receivers, transceivers, transducers, detectors,as well as any devices which can be used to detect images or measure anyone or more parameters related directly or indirectly to one or more ofthe dynamic conditions, including those listed in FIGS. 17A and 17B.

Fluid sensors 2 n represent nanosensors, nanotransmnitters,nanoreceivers, nanotransceivers, nanotransducers, nanodetectors, as wellas any devices which can be used to detect, image or measure one or moreparameters related directly or indirectly to one or more dynamicconditions, including, but not limited to, those listed in FIGS. 17A and17B.

FIG. 42A shows examples of how exemplary sensors A, Bn, 1 and 2 n can becommunicatively coupled or can transmit, receive, transmit and receive,detect and forward data related to, for example, dynamic conditionsand/or other data used as feedback FB_(j)(t). Arrows indicate directionof flow of data or information. Dashed lines are used to indicate allpossible data flow it being understood that actual information flowdepends on the type of sensors. In some embodiments of the invention,sensors may not directly measure, but may instead serve as boosters orrepeaters. For example, in one embodiment of the invention the fluidcontains thousands of nanodetectors and receivers which detect one ormore dynamic conditions and transmit photons of a certain frequencydepending on the presence of certain gases, liquids, solids and/orbiological materials, which in turn can be detected by system sensor A(e.g., photon detector), which in turn puts out a feedback signalFB_(j)(t) to controller 70. Referring to FIG. 42A, this would berepresented by the configuration shown in FIG. 42B.

FIG. 43A shows various possibilities of how six sensors A, Bn, C, 1, 2 nand 3 n can be communicatively coupled or transmit, receive, transmitand receive, detect and forward data related to, for example, dynamicconditions or other data used as feedback FB_(j)(t). Again, in someembodiments of the invention, sensors may not directly measure, but mayinstead serve as boosters or repeaters. Referring to FIG. 43A, thiswould be represented by the configuration shown in FIG. 43B.

According to embodiments of the invention, a receiver (e.g., system orfluid) can have exemplary volumes of less than 1 cm², less than 500 mm²,less than 1 mm², less than 500 μm², less than 100 μm², less than 1 μm²,less than 500 nm², less than 100 nm², less than 1 nm² or the like.According to other embodiments of the invention, at least one receiver(e.g., system or fluid) can have exemplary dimensions of less than or atleast 500 mm along a first direction, 1 mm along a first direction, 500μm along a first direction, 100 μm along a first direction, 1 μm along afirst direction, 500 nm along a first direction, 100 nm along a firstdirection, 50 nm along a first direction, 1 nm along a first direction,0.1 nm along a first direction or the like. In other embodiments, areceiver can have a similar size to a fluid receiver. In otherembodiments, a transmitter, fluid transmitter, a transmitter/receiver,fluid transmitter/receiver can have a similar size to a fluid receiveror receiver. In other embodiments, a sensor (e.g., in a system, fluid,specimen or the like) can have similar sizes to a receiver, transmitter,or transmitter/receiver.

Fluid sensors, system sensors and/or specimen sensors (A, B, . . .A_(n), B_(n), . . . 1, 2, . . . 1_(n), 2_(n), . . . ) discussed herein(e.g., receivers, transmitters, transceivers) may further include probesin accordance with embodiments of the invention. Probes can be used asfluid, system and/or specimen sensors. Probes can characterizebiological activity such as cell activity. For example, biologicalactivity can be observed or detected using activity based probes. Anactivity based probe can form a bond (e.g., irreversible covalent bond)with a desired active biological target (e.g., protein target). Once thetarget is coupled to the probe, the target can be more easily detected,monitored or utilized.

FIG. 44A shows an activity based probe D01 can include an engaging endD05 or warhead, a tag D09 and a connector portion D11 in accordance withone embodiment of the invention. Engaging end D05 can be designed toengage amino acid residue at an active enzyme site D13. Accordingly, thereactivity, polarity, charge, size and structure can set theeffectiveness and selectivity of the probe. Tag D09 is used to detectand/or enrich the active target. Tag D09 can include a radioactivemolecule, fluorescent or the like. Tag D09 can be, for example, abiotin, a radioactive molecule, or a fluorescent molecule such asfluorophore or 125I. Connector portion D11 is a molecular chain whichcan reduce interference between tag D09 and engaging end D05 as well asto assist in selecting the probes target D13 can be, for example, apeptide, alkyl polyether or the like. Engaging end D05 can be aphosphonate, fluorophosphonate, epoxyketone or the like.

FIG. 44B shows a probe D51 which is smaller and preferably significantsmaller than target D53. Here, probe D51 also has an engaging end D05and a connector portion D11 but with tag D09 replaced with tag D55 whichincludes a drug or pharmacological agent or any small or large molecules(for example, see FIG. 17B). Hence probe D51 can provide a deliverymechanism such as a drug delivery mechanism to tubular structures inaccordance with embodiments of the invention. In an alternativeembodiment of the invention, tag D55 can be a combination of tag D09 ofFigure DA and a drug, pharmacological agent or any other small or largemolecules, in which case probe D51 can serve both as a deliverymechanism and a sensor for the tubular structure or specimen and/or tofluid materials in fluids described herein and/or to flow loop fluids insystems such as systems 1, 1101 in accordance with other embodiments ofthe invention.

FIG. 44B shows yet another embodiment of probe D51 (dashed lines) inwhich a second tag D57 is attached with a second connector portion D61in accordance with another embodiment of the invention. Hence, probe 51with tag D55 functions in a similar manner to that described above withrespect to Figure DA, and probe 51 with tag D57 functions as a deliverymechanism as described herein.

FIG. 45A shows tubular structures 1112 which are permeable orsemipermeable to fluid materials of any kind, and shown as permeabletubular structures 1152, in accordance with embodiments of theinvention. Permeable tubular structures 1152 allow for the migration,flow and/or diffusion of fluid and/or any fluid materials, such asparticles, sensors, or molecules as described herein, examples of whichare listed in FIGS. 17A and 17B. Hence, measurement of the amount, flow,velocity of fluid or fluid material corresponds to measurement of typesof dynamic conditions, examples of which are shown in FIGS. 17A and 17B.

The direction of velocity and flow can include measurement of adirectional dynamic condition g(t) having a component in the verticaldirection, as well as measurement of nondirectional dynamic conditionsg(t), such as amounts of fluid or fluid material. System and/or fluidsensors can be used to measure these types of dynamic conditions, inaccordance with embodiments of the invention.

FIG. 45B shows other types of tubular structures 1112, which are porousor semi porous tubular structures 1155, in accordance with embodimentsof the invention. Again, tubular structures 1155 allow for themigration, flow and/or outfusion of fluid and/or fluid material asdescribed herein, examples of which are shown in FIGS. 17A and 17B.Hence, measurement of the amount, flow, velocity or other dynamiccondition of fluid or fluid material constitutes measurement of types ofdirectional dynamic conditions g(t) and/or measurement of typesnon-directional dynamic conditions g(t), as shown in FIGS. 17A and 17B.Again, directional dynamic variables may include a nonzero component inthe radial direction.

FIG. 45C shows other types of tubular structures 1112 which areelectrospun tubular structures 1157, preferably made of fibrin in amanner such as that described, for example, in U.S. Pat. Nos. 6,592,623and 6,787,357, the contents of which are incorporated herein byreference. Electrospun tubular structures 1157 can be permeable and/orporous.

FIG. 46 shows other types of tubular structures 1112, which aremicrogrooved tubular structures 1252. Microgrooved tubular structures1252 have depths of 50 nm and 700 nm, can have grooves with widths ofbetween 40 nm and 2000 nm and preferably 70 nm and 1400 nm, and pitchbetween approximately 200 to approximately 5000, and preferablyapproximately 400 to approximately 4000 depending on the class ofdynamic condition (FIG. 18) which it will be subjected to, as well asthe type of cells and/or coatings which might be applied to it.

Tubular structures 1112 can be combinations of two or more of the abovetubular structures, such as two or more tubular structures 1112 in FIGS.11, 12, 13A, 13B, 14, 14, 16, tubular structures 1152, 1155, 1157 and1252 and specimens 12 in FIGS. 1A, 2A-2E, 3A-3D, 5A-5D and 6A-6E. Hence,a tubular structure could be a combination of a porous tubular structure1155 (FIG. 45B) and a microgrooved tubular structure 1252 (FIG. 46).

Second order dynamic conditions can include any type of dynamicconditions such as those listed in FIGS. 17A and 17B and any othersknown in the art. Second order dynamic conditions are localized dynamicconditions produced by systems discussed herein, such as systems 1 and1101, in conjunction with tubular structures having perturbed physicalcharacteristics and/or properties when those systems operate to providea given set of global dynamic conditions and/or dynamic conditions atone or multiple regions A in accordance with embodiments of theinvention. When these systems operate to provide a given set of globaldynamic conditions and/or known types of dynamic conditions at one ormultiple regions A in accordance with embodiments of the invention.

Such perturbed tubular structure characteristics include, for example,shape, structure, porosity, permeability, dimensions, thickness,stiffness, elasticity, ridges, localized stiffness and elasticity,protrusions, bumps, rigid full rings or rigid partial rings, expandablefull rings or partial rings with known elasticities, as well as rigidfull sleeves or rigid partial sleeves, or full or partially expandablesleeves with known elasticity. Other perturbed tubular structurecharacteristics can include coatings with altered coefficients offriction, smoothness and/or roughness of the inner surface of thetubular structure, and the spatial frequencies of any repeatingstructure perturbation, such as small bumps, rings, grooves, sleeves,shapes and so forth, examples of which will be discussed with respect toFIGS. 47A-47H.

These dynamic conditions are referred to herein as second order dynamicconditions because they result the use of the perturbed tubularstructures in systems which operate to provide predetermined or knownglobal dynamic conditions of one or multiple regions A. Second orderdynamic conditions can be used, for example, to effect additional setsof dynamic conditions which might be present in vivo for healthy,diseased, or other in vivo tubular structures. Second order dynamicconditions can also be used, for example, to create additional sets ofdynamic conditions or other non-biological situations, as well asdynamic conditions useful for training and testing, or growing tubularstructures, samples of which are shown in FIG. 18.

All tubular structures discussed throughout can include biologicalmaterial, such as cells, etc., can include a hybrid of biologicalmaterial and non-biological material, synthetic or non-syntheticnon-biological material or completely biological material, such as veinsor arteries or tissues, or organs and so forth as described herein.

Variations in cross-sectional area along the z direction correspond tovariations in D as broadly defined herein. Hence, tubular structurealong Z can be represented by D(z). The z axis can represent anapproximately straight line along the direction of the mean pulsatoryflow in tubular structures, in accordance with embodiments of theinvention. Alternatively, the Z direction can represent a line fiatfollows approximately along the center of each cross-sectional area of atubular structure, according to other embodiments of the invention.

Tubular structures also include, for example, biological ornon-biological or hybrid biological and non-biological tubularstructures which have been in any way slightly, moderately orsubstantially modified as a result of being subjected to one or moresets of dynamic conditions and second order dynamic conditions for anamount of time sufficient to yield any such slight, moderate orsubstantial modifications of the tubular structure itself. Again,tubular structures can have perturbed physical characteristics and/orproperties which immediately yield desired dynamic conditions, includingsecond order dynamic conditions, once placed in systems described hereinwith the appropriate global dynamic conditions, including systems 1 and1101, according to embodiments of the invention.

FIGS. 47A-47H show examples of tubular structures or specimens 12, 1112which can be used to effect second order dynamic conditions. Again, asdiscussed herein, these tubular structures and specimens, as well as allother specimens and tubular structures including, for example, anyone ormore combinations of those shown in FIGS. 1A, 11, 12, 13A, 13B, 14, 15,16, 36, 38, 42, as well as any portion or section of systems, such assystems 1, 1101 which can pass fluid from one location to another asdefined herein (see FIGS. 11 and 12), can be porous, non-porous,permeable or non-permeable or any hybrid thereof biological,non-biological or any hybrid thereof, multilayered, multi-channeled ormultiple branched and any combination of these and one or multipletubular structures shown in FIGS. XA-XH. Again, tubular structures canhave perturbed physical characteristics and/or properties which nearlyimmediately yield the desired dynamic conditions including second orderdynamic conditions once placed in systems 1, 101 and/or 1101 with theappropriate global dynamic conditions.

FIG. 47A shows a tubular structure 12, 1112 with varying diameter Dalong a z direction which, in accordance with embodiments of theinvention, corresponds to variation of the cross-sectional areas oftubular structures along the z direction. Again, diameter D, and henceat cross-sectional areas at Z₁ . . . Z_(n) of tubular structures asdefined herein can include FIGS. 12, 13A, 13B and 17. Hence, D₁ maycorrespond to a circular cross-sectional area and D₂ may correspond toan ovular cross-sectional area. Generally, D₁ and D₂ can be, forexample, any cross-sectional area including those, for example, in FIG.12, and the transition from D₁ to D₂ along the z direction can be anyseries of cross-sectional areas. FIGS. 47B, 47C, 47D show more examplesof tubular structures in which D₁ and D₂ might be approximately thesame, but the transition of cross-sectional areas varies along the zdirection by becoming larger then smaller (FIG. 47B) or becoming smallerthen larger FIG. 47C).

FIG. 47D shows another example of tubular structures withcross-sectional variations along the z direction. If z represents theapproximate center of cross-sectional areas D(z), z=z₁-z_(n), then thefirst cross-sectional area D(z₁) might represent a circle having aradius of r₁. D(z₂) might represent a cross-sectional area which isovular on the top half with a major axis radius r₂, and circular on thebottom half still with the radius r₁, where r₂>r₁. Cross-sectional areaD(z₃) might be ovular with a major axis of r₃ and a minor axis r₄, wherer₃>r₄ and, for example, r₄>r₁.

FIG. 47E shows a tubular structure with grooves 2500 according toadditional embodiments of the invention. FIG. 47E has grooves angledwith respect to the Z direction. Here, the grooves are consideredcompletely aligned with the z direction if they run approximatelyparallel to the z direction. It should be understood that grooves can beany grooves, microgrooves, ridges, indentations and can be on the innerdiameter, the outer diameter (to effect, for example, a particularflexibility of elasticity) of the tubular structure and/or within thewall of one or more layers of the tubular structure, according toembodiments of the invention. Grooves, as used herein, include thepresence and/or absence of any biological and/or non-biologicalmaterials including, but not limited to, materials used or present inany tubular structures as defined herein. Accordingly, grooves can betroughs having a desired cross-sectional shape such as a “V” shape, semior partially circular or ovular shape, rectangular shape and so forth.Variations in the depth, width, length, direction, shape and/orperiodicity (for example, distance between grooves) can produce or alterthe resulting second order dynamic conditions for a given set of globaldynamic conditions and/or dynamic conditions at region(s) A.

FIG. 47F shows a tubular structure with projections on an interiorsurface according to additional embodiments of the invention. Bumps 2515can provide fluid perturbations as desired or that provide selectedempirical results. Bumps 2515, as used herein, include the presenceand/or absence of any biological and/or non-biological materialsincluding, but not limited to, materials used or present in any tubularstructures as defined herein. Bumps can have a desired cross-sectionalshape such as a circular shape, semi or partially circular or ovularshape, rectangular shape and so forth. Variations in the depth, width,length, direction, shape and/or periodicity (for example, distancebetween bumps) can produce or alter the resulting second order dynamicconditions for a given set of global dynamic conditions and/or dynamicconditions at region(s) A.

FIG. 47G shows a tubular structure 12, 1112 in accordance with anotherembodiment of the invention. Tubular structure 12, 1112 has a partialring 4001, a full ring 4003, a full sleeve 4005, a partial sleeve 4007and a patch 4009 attached and/or coupled to tubular structure 12, 1112and/or fabricated into tubular structure 12, 1117. Rings 4001 and 4003,sleeves 4005 and 4007 and patch 4009 can be ridged or flexible toprovide known variations in flexibility and elasticity of the walls oftubular structure 12, 1112 along the z axis. Such known variations inflexibility and elasticity yield sets of dynamic conditions includingsecond order dynamic conditions at tubular structure 12, 1112 whichcorrespond to certain desired dynamic conditions when used in systemsherein in accordance with embodiments of the invention such as systems1, 1101.

As discussed above, FIGS. 47A-47G are tubular structures which can beformed outside systems 1, 1101 or tubular structures that are formed,trained and/or grown after being subjected to predetermined dynamicconditions, including global and/or dynamic conditions at regions A fora predetermined amount of time. Hence, an initial tubular structure withinitial perturbations and predetermined variations in shape D(z), candevelop a desired shape and desired perturbations to effect the desiredsecond order dynamic conditions as a result of the growth and/or furtherdevelopment of grooves, varying elasticity and/or any otherperturbations of the tubular structure due to the growth and/or trainingof biological material on and/or the tubular structure.

Also, tubular structures can be a combination of one or more tubularstructures with biological or non-biological materials which can beapplied to the inner surface and/or the outer surface of the tubularstructures. In addition, tubular structures include all of the abovecombinations which have been placed in the systems described herein andallowed to develop, grow under desired dynamic conditions for a desiredlength of time. Such tubular structures are said to have been tubularstructures as shown in FIG. 18 under training/testing dynamic conditionsand, in particular, non-in vivo bio training/testing conditions. In vivodynamic conditions might also serve as training/testing conditions aswell as combinations of in vivo dynamic conditions and training/testingdynamic conditions.

Tubular structures can be slightly, partially, substantially orcompletely trained in the absence of any biological materials as well.This might include subjecting tubular structures to training by thesystems, in accordance with other embodiments of the invention in orderto prepare them or alter them or test them for a particular use.

Referring back to FIG. 18, classes of dynamic conditions can besubdivided into areas or locations at which the dynamic condition mightoccur. Hemodynamic conditions can, for example, be divided intohemodynamic conditions experienced by arteries in the upper leg orarteries in the lower leg, veins in the upper legs or veins in the lowerlegs, arteries in the arms, veins in the aims, pulmonary arteries,arteries in the neck and so forth. Hybrid tubular structures 1112 caninclude endothelial cells grown under dynamic conditions which includecombinations of types of dynamic conditions (FIGS. 17A and 17B) for aparticular class of dynamic conditions, e.g., a particular artery in theupper leg. Resulting morphology and functionality of the endothelialcells grown (from stem cells) and/or trained under upper leg arteryhemodynamic conditions of a particular mammal will be different than themorphology and functionality of a particular vein grown and/or trainedunder hemodynamic conditions experienced by the particular vein in theparticular mammal (see, for example, David G Harrison “The shear stressof keeping arteries clear” Nature Medicine, Vol. 11, No. 4, April 2005,pp. 375-376; James N. Topper et al., “Blood flow and vascular geneexpression; fluid shear stress as a modulator of endothelial phenotype”Molecular Medicine Today, January 1999, pp. 40-46; Ulf Landmesser, MD etal. “Endothelial Function, A Critical Determinant in Atherosclerosis”American Heart Association, Jun. 1, 2004, pp. II-27-II-33; Edward M.Boyle, Jr., MD et al. “Atlerosclerosis” 1997 by The Society of ThoracicSurgeons, pp. S47-S56; Peter F. Davis, et al. “Spatial Microstimuli inEndothelial Mechanosignaling” Circulation Research Mar. 7, 2003, pp.359-370; Shu Chien “Molecular and mechanical bases of focal lipidaccumulation in arterial wall” Progress in Biophysics & MolecularBiology 83 (2003), pp. 131-151; Michael B. Dancu et al. “AsynchronousShear Stress and Circumferential Strain Reduces Endothelial NO Synthaseand Cyclooxygenase-2 but Induces Endothelin-1 Gene Expression inEndothelial Cells” Arterioscler Thromb Vasc Biol., November 2004, pp.2088-2094; Ruey-Bing Yang et al. “Identification of a Novel Family ofCell-surface Proteins Expressed in Human Vascular Endothelium” TheJournal of Biological Chemistry, Vol. 277, No. 48, Issue of Nov. 29,2002, pp. 46364-46373; Filomena de Nigris et al. “Beneficial effects ofpomegranate juice on oxidation-sensitive genes and endothelial nitricoxide synthase activity at sites of perturbed shear stress” PNAS, Mar.29, 2005, vol. 102, no. 13, pp. 4896-4901; and Ralph L. Nachman et al.“Endothelial cell culture: beginnings of modern vascular biology” TheJournal of Clinical Investigation, vol. 114, no. 8, October 2004, pp.1037-1040, which are all hereby incorporated by reference in theirentirety).

As used herein, fluids passing through pressure flow system, tubularstructures specimen holders and/or grafts or the like have beenvariously described. However, fluids are not intended to be so limited.For example, fluids used in embodiments or as embodiments can includefluid materials such as liquids, solids, gases and/or miscellaneousitems, individually or in various combinations, concentrations ormixtures. Exemplary fluids can include fluid materials such as cells,bacteria, minimum essential Eagles medium, growth factor, celldifferentiating small molecule, cell differentiating biologics, cellculture medium or the like. Exemplary liquids can include plasma,saline, blood, water, cell culture medium, fetal bovine serum (FBS),bovine serum albumin (BSA), cerebral spinal fluid or the like. Fluidmaterials can include solids such as hormones, proteins, viruses,lipids, peptides, nucleotides, glycols, antibiotics, pharmacologicalagents, transmitters, receivers, transmitter/receivers, fluidnanoparticles, free electrons, minerals, iron zinc, copper, magnesium,calcium or the like. Exemplary gases can include oxygen, nitric oxide,carbon dioxide, carbon monoxide or the like.

Systems herein including systems 1 and 1101 can be used to model orsimulate. According to one embodiment, systems 1 and 1101 together withperturbed tubular structures can model pathology or the departure ordeviation from a normal condition at the tubular structure. This caninclude anatomic or functional manifestations of a disease (orstructural and functional changes in cells, tissues and organs thatunderlie disease). Systems 1 and 1101 can model pathologies withinvarious classes of dynamic conditions (e.g., see FIG. 18) using at leastone and typically multiple types of dynamic conditions (e.g., see FIGS.17A and 17B) according to embodiments of the invention. Accordingly,embodiments of systems 1 and 1101 can be used to determine or evaluatedynamic, static, time dependent, non-linear or changing behaviors.

The functional phenotype of vascular endothelium can be responsive(e.g., dynamically) to an array of physiological and pathophysiologicalstimuli al of which represent types of dynamic conditions as per FIGS.17A and 17B. Such stimuli can include biochemical substances such asinflammatory cytokines, growth factors, circulating hormones andbacterial products. In addition, endothelium is exposed to a number ofbiomechanical stimuli resulting from the pulsatile flow of blood withinthe branched vascular tree including frictional forces, fluid shearstresses, cyclic strains (stretch) and hydrostatic pressures or the like(yet additional types of dynamic conditions as per FIGS. 17A and 17B).

Shear stress stimulates a myriad of intracelluar events (e.g.,intracellular signaling events) in endothelial cells which alsorepresent types of dynamic conditions of FIGS. 17A and 17B. Some ofthese events, such as changes in intracellular calcium, proteinphosphorylation and acute stimulation of nitric oxide production, occurwithin seconds after the onset of shear and other changes such as cellshape and gene expression, occur over hours to days.

Embodiments of the system can model vascular diseases using varioustypes of dynamic conditions. For example, the interplay betweenhemodynamic stimuli and the functional phenotype of endothelium canaffect a variety of vascular diseases.

One exemplary set of biomechanical and intracellular signaling dynamicconditions in endothelial cells can be for atherosclerosis.Atherosclerosis is a progressive disease, and changes within thearterial endothelium, such as an increased permeability to lipoproteins,endothelial cell damage and/or repair, and the expression of leukocyteadhesion molecules can be demonstrated in the atherosclerotic process.Interactions between apoptosis signaling kinase 1 (ASIK1), Txnip, whichis a molecule whose levels correlate with the degree of shear stress,and thioredoxin in endothelial cells can be related to shear stress.Txnip binds to catalytic cysteines of thioredoxin to reduce thioredoxinactivity and its ability to bind to ASK1. See, for example, Blood Flowand Vascular Gene Expression: fluid shear stress as a modulator ofendothelial phenotype, Topper, J. N., and Gimbrone Jr., M. A., MolecularMedicine Today, January 1999, pp. 40-46; the contents of which areincorporated herein by reference.

Additional events that can be modeled include interactions ofendothelial cells and the types of dynamic conditions measured and/orcontrolled by systems 1 and 1101 can include nitric oxide production,enhanced expression of antioxidant enzymes like superoxide dismutase andglutathione peroxidase or glutathione.

In one embodiment of the invention, a specimen 12 such as tubularstructure 1112 with endothelial cells is placed in a specimen holder 10in pressure flow loop subsystem 1105, while the dynamic condition ofshear stress is controllable varied and detection of ASK1, thoioredoxinand Txnip are monitored by systems, specimens or fluid sensors. In theendothelial cells, shear stress associated to thoioredoxin can affectactivation of ASK1. In the absence of shear stress, thioredoxin is boundby Txnip and maintained in an inactive state, which leads to increasedactivation of ASK1. This leads to increased expression of the vascularcell adhesion molecule 1 (VCAM1), which promotes leukocyte adhesion,inflammation and atherosclerosis. For example, cytokine TNF-α can leadto phosphorylation and activation of ASK1, and the activated ASK1activates downstream MAP kinases and ultimately p38 and Jun-terminalkinase (JNK), which increases VCAM1.

However, in the presence of shear stress, Txnip can be reduced,liberating thioredoxin and leading to increased binding of thioredoxinto ASK1 and inhibition of ASK1 (e.g., less ASK1 activation by TNF-α).Thus, shear stress can be controllably set between 0 and a maximum valuein a series of steps while data is collected according one embodiment.Then, results can be related to corresponding levels of theintercellular activity by, for example, controller 70 or 1103.

As described above, such interrelationships between dynamic conditionsillustrate exemplary modeling targets for disclosed embodiments.Exemplary modeling that can be performed by system embodiments ormodeling embodiments are shown in FIG. 40.

Similarly, embodiments of systems and methods described herein withrespect to FIGS. 1-47 and be used for testing/training activities.Embodiments can be used for exemplary dynamic conditions related totesting and training as described herein.

FIG. 48 shows steps of a preferred method for producing dynamicconditions at regions A, as well as second order dynamic conditions at aspecimen or tubular structure 12 or 1112. The method starts at step4801, at which dynamic conditions g_(j)(t) (see FIG. 17), includingsecond order dynamic conditions and mean dynamic conditions, aremeasured in vivo.

Step 4805 involves selecting a tubular structure used to train system 1,1101 that yields a set of dynamic conditions closest to the dynamicconditions g_(j)(t)s measured in step 4801. Step 4810 involves selectinginitial global dynamic conditions and/or conditions at one or moreregions A for system 1, 1110 based on the mean of the measured dynamicconditions g_(j)(t).

Step 4815 involves measuring a first set of resulting dynamic conditionsg_(j)(t) at the known stable tubular structure. Any of the methods andsystems described herein (for example, sensors A, B, Cn, Dn . . . and/or1, 2, 3 n, 4 n . . . ), as well as any other methods and systems knownin the art, can be used to directly and/or indirectly measure theresulting dynamic conditions.

Step 4820 involves perturbing the stable tubular structure in a manner,for example, as discussed above in connection with FIGS. 45A-45C, 46,and 47A-47H, or as otherwise discussed herein. This may involvereplacing the stable tubular structure with a second perturbed tubularstructure. The stable tubular structure is preferably perturbed in amanner which will change the set of resulting dynamic conditions,including resulting second order dynamic conditions, to values that arecloser to the measured set of dynamic conditions. For example, a rigidfull ring (FIG. 47H) can be used to alter the resulting dynamicconditions and second order dynamic conditions.

At step 4825, it is determined whether the resulting set of dynamicconditions measured at step 4801 is sufficiently close to the measuredset of dynamic conditions or a desired set of dynamic conditions. If itis, the method ends. If not, then the method jumps back to step 4715.

In the method embodiment above, it is assumed that a selected class ofdynamic conditions (e.g., FIG. 18) was determined prior to step 1.Further, steps 2 and 3 above presumes that a plurality of systems havingdifferent components have been trained using a plurality of initialtubular structures. The initial systems could include systems such assystems 1, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 1101.Further, the known stable initial tubular structures could be anytubular structures such as shown in FIGS. 5A-5D, 45A-45C, 46 and47A-47H. In addition, perturbing the known stable tubular structure canbe considered to include another embodiment modifying the global dynamicconditions in the pressure flow loop subsystem (e.g., pressure flow loopsubsystem 1105) to generate dynamic conditions at a tubular structuremounted therein or one or more regions A on such a tubular structure.

As discussed above, communications between the controller 1105 andsensors (e.g., A, B, . . . ; 1, 2 . . . ) can include communicationsbetween sensors in the fluid (represented herein by numbers such as 1,2, . . . ) in the system (represented herein by letters A, B, . . . )and/or in the specimen or tubular structure represented either bynumbers 1, 2, . . . , or letters A, B, . . . , depending on whether theywere part of the system or part of the fluid that can individuallytransmit (t), receive (r) or transmit and receive (tr). System sensor insystem 1101 can be in the pressure flow loop subsystem 1105 includingcomponents thereof and/or the specimen 10 and directly or indirectlycoupled to each other and to system 1101 including controller 70 todetect dynamic conditions (e.g., FIGS. 17A and 17B). Sensors in thefluid or fluid sensors are referred to herein from time to time as fluidtransmitters, fluid receivers, fluid transmitter/receivers and/or fluiddetectors. For example, such fluid sensors can include nanoparticlessuch as nanosensors and/or mems sensors which are indicated as 1 n, 2 n. . . rather than 1, 2, . . . . Exemplary transmitter, receiver ortransmitter/receiver nanoparticles (system, fluid and/or specimen) caninclude a nanotransmitter, nanoreceiver or a nanosensor or ananotransmitter/receiver.

A transmitter/receiver (system, fluid and/or specimen) is capable ofreceiving information from another transmitter/receiver (system, fluidand/or specimen) a transmitter (system, fluid and/or specimen), atransmitting nanoparticle (system, fluid and/or specimen) a transmittingsensor (system, fluid and/or specimen) or the like, and/or combinationsthereof. The transmitter/receiver (system, fluid and/or specimen) iscapable of sending information to another transmitter/receiver (system,fluid and/or specimen), a receiver (system, fluid and/or specimen), areceiving nanoparticle (system, fluid and/or specimen), a receivingsensor (system, fluid and/or specimen), or the like and/or combinationsthereof. Further, a first transmitting or receiving sensor and/or memsor nanosensors (system, fluid and/or specimen) or a plurality of firsttransmitting or receiving sensors and/or mems or nanosensors each can becapable of sending or receiving information to/from at least one secondtransmitting or receiving sensors and/or mems or nanosensors (system,fluid and/or specimen) or vice versa.

Such transmitting or receiving relationships can exist for a transmitteror a receiver. A transmitter (system, fluid and/or specimen) is capableof sending information to a transmitter/receiver (system, fluid and/orspecimen), a receiver (system, fluid and/or specimen), a receivingnanoparticle, a sensor or the like, a plurality of the same, individualitems above or combinations thereof. Further, the transmitter is capableof sending information to one or more second receivers (e.g., system orfluid) and/or one or more third receivers (e.g., system or fluid).Similarly, a plurality of first transmitters (e.g., system or fluid) caneach be capable of sending information to a single or designatedreceiver or sensor.

A receiver (e.g., system or fluid) is capable of receiving informationfrom a fluid transmitter/receiver, a transmitter/receiver, a fluidtransmitter, a transmitter, a transmitting nanoparticle, a sensor, afluid sensor or the like, a plurality of the same, individual itemsabove or combinations thereof. Further, the receiver is capable ofreceiving information from one or more second transmitters (e.g., systemor fluid) and/or one or more third transmitters (e.g., system or fluid).A plurality of first receivers (e.g., system or fluid) can each becapable of receiving information from a single or designated transmitteror sensor.

A transmitter/receiver nanoparticle (e.g., system or fluid) is capableof sending and/or receiving information to/from a transmitter/receiver,a fluid transmitter/receiver, a transmitter/receiver nanoparticle, atransmitter, a fluid transmitter, a receiver, a fluid receiver, ananoreceiver, nanotransmitter, a fluid sensor, a sensor or a nanosensoror the like, a plurality of the same, individual items above orcombinations thereof. A first transmitting and/or receiving nanoparticle(e.g., system or fluid) is capable of sending or receiving informationto/from one or more second transmitting and/or receiving nanoparticlesand/or one or more third transmitting and/or receiving nanoparticles ormore. A plurality of first transmitter/receiver nanoparticles is capableof sending or receiving information to/from a single or designatedtransmitter/receiver nanoparticle.

Such transmitting or receiving relationships can exist for ananotransmitter or a nanoreceiver. A nanotransmitter (e.g., system orfluid) is capable of sending information to a nanotransmitter/receiver,a fluid nanoreceiver, a receiver, a fluid receiver, a fluid sensor, asensor, a nanosensor, a nanoreceiver or the like, a plurality of thesame, individual items above or combinations thereof. Further, a firstnanotransmitter is capable of sending information to one or more secondreceiver nanoparticles (e.g., system or fluid) and/or one or more thirdreceiver nanoparticles (e.g., system or fluid) or more. Similarly, aplurality of first nanotransmitters (e.g., system or fluid) can each becapable of sending information to a single or designated nanoreceiver orsensor (e.g., system or fluid).

A nanoreceiver (e.g., system or fluid) is capable of receivinginformation from a fluid nanotransmitter/receiver, ananotransmitter/receiver, a transmitter/receiver, a fluidtransmitter/receiver, a fluid transmitter, a transmitter, ananotransmitter, a sensor or the like, a plurality of the same,individual items above or combinations thereof. A first receivernanoparticle is capable of receiving information to/from one or moresecond transmitter nanoparticles (e.g., system or fluid) and/or one ormore third transmitter nanoparticles (e.g., system or fluid) or more. Aplurality of first nanoreceivers (e.g., system or fluid) can each becapable of receiving information from a single or designatednanotransmitter or a sensor (e.g., system or fluid).

Exemplary communications between sensors can be supported by describedembodiments. For example, a transmitter/receiver (or a separate transmitsensor and receiver sensor) is capable of transmitting information to ortransmitting/receiving information to/from a first transmitter/receiver(or a plurality of first transmitter/receivers) and a secondtransmitter/receiver (or a plurality of second transmitter/receivers) iscapable of receiving information from the first transmitter/receiver (orthe plurality of first transmitter/receivers) and transmittinginformation to the transmitter/receiver. In this case, thetransmitter/receivers (e.g., system or fluid) can be transmitters orreceivers and can be nanoparticles (e.g., system or fluid) orcombinations thereof.

In another example supported by disclosed embodiments, atransmitter/receiver is capable of receiving information from ortransmitting/receiving information to/from a first transmitter/receiver(or a plurality of first transmitter/receivers) and a second fluidtransmitter/receiver (or a plurality of second transmitter/receivers) iscapable of transmitting information to the first fluidtransmitter/receiver (or the plurality of first transmitter/receivers)and receiving information from the transmitter/receiver. In this case,the transmitter/receivers (e.g., system or fluid) can be transmitters orreceivers and can be nanoparticles (e.g., system or fluid) orcombinations thereof.

A transmitter or receiver is capable of transmitting information to ortransmitting/receiving information to/from a plurality of firsttransmitters or receivers and a plurality of second transmitters orreceivers is capable of respective communications (e.g., 1-to-1,many-to-1, 1-to-many, many-to-many, 1-to-all, all to-1, all-to-all, orthe like) with each of the first transmitters or receivers to thentransmit information to the transmitter or receiver. Atransmitter/receiver is capable of receiving information from ortransmitting/receiving information to/from at least one first fluidtransmitter/receiver, and a plurality of second fluidtransmitter/receivers are capable of receiving information from thefirst fluid transmitter/receiver and transmitting information to atleast one third fluid transmitter/receiver that can transmit informationto the transmitter/receiver or vice versa. Further, a system 1, 1101could include four or more plurality of sensors in respectivecommunications (e.g., 1-to-1, many-to-1,1-to-many, many-to-many,1-to-all, all to-1, all-to-all, or the like) with subsets orcorresponding ones of each other pluralities to transmit respectiveinformation f_(i)(t), f_(j)(t) or FB_(j)(t) therein. Again, sensors(e.g., system or fluid) can be nanoparticles (e.g., system or fluid) orcombinations thereof.

Applications of Systems

Applications for embodiments of the system broadly described herein arenumerous and varied. Although the exemplary discussion set forth hereinhas focused mainly on biological applications for embodiments of thesystem, and more particularly, on hemodynamic forces which act on bloodflowing through blood carrying vessels, it is well understood thatembodiments of the system may be used to reproduce any dynamic pressureand flow environment which would benefit from the ability toindependently control pressure and flow, including both biological andnon-biological applications. For example, embodiments of the system maybe used to produce a biological environment which would simulate anumber of other in vivo conditions. These conditions may include, forexample, pressure and flow conditions within joints or on variousbone/tendon/musculature structure. Embodiments of the system may also beused to reproduce conditions within, for example, the stomach,intestines, esophagus, lungs, or sinus cavities, or any other suchdynamic in-vivo-environment in which such a reproduction of actualconditions would prove beneficial. Embodiments of the system may, withthe proper cellular structure, seeding, and corresponding media, be usedto initiate and grow replacement bone/cartilage/organ structure and thelike.

Non-Biological Applications

In addition to these dynamic in-vivo conditions, certain systems asembodied and broadly described herein may also be used to reproducedynamic pressure and flow environments to which non-biological elementsare subjected during operations. Such non-biological applications mayinclude, for example, dynamic pressure and flow through rigid/flexiblepipes/tubes in a whole host of different systems. These systems mayinclude, but are not limited to, for example, petroleum pipelines, fuelflow lines in a variety of different systems, hydraulic systems,lubrication systems, fluid lines in manufacturing facilities, andespecially those under pressure, drainage systems and related stormwater and sewage treatment systems, and any other suchpractical/industrial application which would stand to benefit from sucha modeling of actual conditions.

Biological Applications

Applications in the pharmaceutical, biotechnology, life science,academic, and research industries for a system as embodied and broadlydescribed herein include, but are not limited to, therapeutic screening& testing and discovery & development of drugs, active biomolecules,regenerative medicines, medical devices, cell & tissue devices ortherapies, drug delivery systems, personalized medicine,genomics/proteomics, small chemical, biologics, and the like.Embodiments of the system can be adapted to serve as a model forcardiovascular and related pathologies such as, for example, cancer ordiabetes, via simulation of pathologic (or non-pathologic) hemodynamicsthat induce a consequent pathologic (or non-pathologic) response andphenotype on vascular cells as well as other cells. Therapies can thenbe designed, developed, and tested against the pathologic model. Thisdynamic cell and tissue culture environment captures in vivo phenotype,function, and physiology more closely than traditional static cultures.

Embodiments of the system can enhance and reduce therapeutic discoverytimes by providing a cost-effective platform to perform typical andnovel cell, molecular biological, and pharmacological research anddevelopment. Embodiments of the system not only provides a means ofstudying hemodynamics in normal and diseased states, but can also beused for tissue engineering and regeneration, such as, for example, totest or train the function of bypass vessels prior to coronary bypasssurgery or peripheral arteries or AV-shunts, or to investigatecryopreserved vessels or for research or medical use. Exampleapplications include atherosclerosis, plaques (vulnerable plaque,protruding, calcified, soft, etc.), inflammation (i.e. leukocyteadhesion), restenosis, cancer (metastasis-tumor spreading to othertissues, extravasation-tumor and leukocyte adhesion, and the like), anddiabetes (retinopathy, blindness, and the like).

Embodiments of the system can also provide essential capabilities andhigh-throughput abilities to the pharma/biotech industries, cell-basedscreening and testing, drug discovery and development, and the like.Cell-based assays inherently evaluate test compound activity in abiologically relevant context, with the added potential for extractionof high information content. Embodiments of the system can be used tosystematically screen and test vast numbers of compound combinations,testing their effects using cell-lines or primary-cell or stem cell orpatient specific stem cell cultures to allow interaction with complexbiological pathways that cannot be replicated in a cell-free assay.Other multiple cell or tissue types can be added to certain systems suchas hepatocytes, renal, cardiac, etc. for various purposes such asproviding a test or growth environment that is representative of in-vivoenvironments.

Embodiments of the system may be used in identifying potential chemicalinhibitors or activators of enzymes, receptors, or any proteins whichhave effects upon cell phenotype. One method generally employs two celllines, preferably alike except for their expression (production) of theprotein of interest at different levels (and any further differencesnecessitated by that difference in expression). Inhibitors or activatorsare identified by their greater effect on the phenotype of the higherproducing cell line.

Any phenotypic characteristic of the cell which is affected byexpression of the protein of interest, other, of course, than the levelof the protein itself, may be assayed. The phenotypic characteristic ispreferably a “cultural” or “morphological” characteristic of the cell.For purposes of this application, these terms may be defined as follows.

Cultural characteristics include, but are not limited to, the following:the nutrients required for growth; the nutrients which, though notrequired for growth, markedly promote growth; one or more physicalconditions (temperature, pH, gaseous environment, osmotic state, andanchorage dependence or independence) of the culture which affectgrowth; and the substances which inhibit growth or even kill the cells.

Morphological characteristics include, but are not limited to, thefollowing: the size and shape of cells; their arrangements; celldifferentiation; and subcellular structures.

Where the protein of interest is implicated in tumorigenesis or relatedphenomena, the characteristic observed is preferably one related tocellular growth control, differentiation, de-differentiation,carcinogenic transformation, metastasis, tumorigenesis, or angiogenesis.

Phenotypic changes which are observable with the naked eye are ofspecial interest. Changes in the ability of the cells to grow in ananchorage-independent manner, to grow in soft agar, to form foci in cellculture, and to take up selected stains, for example, are particularlyappropriate phenomena for observation and comparison.

The higher producing cell line is preferably obtained by introducing agene encoding the Protein of Interest (POI) into a host cell or, if anative protein of the cell, by introducing a promoter into the cellulargenome upstream of and operatively linked to the POI. The gene may be aone isolated from the genome of an organism, a cDNA prepared from anmRNA transcript isolated from an organism, or a synthetic duplicate of anaturally occurring gene. It may also have a sequence which does notoccur exactly in nature, but rather corresponds to a mutation (single ormultiple) of a naturally occurring sequence (also referred to as a“wild-type sequence”). No limitation is intended on the manner in whichthis mutated sequence is obtained.

The gene is preferably operably linked to a promoter of gene expressionwhich is functional in the host, such that the corresponding POI isstably “overproduced” in the recipient cells to differing degrees. Thepromoter may be constitutive or inducible. By “overproduced”, it ismeant that the POI is expressed at higher levels in the geneticallymanipulated cell line than in the original cell line. This allows one togenerate cell lines which contain (or secrete) from as little as a fewfold to more than 100-fold elevated levels of the POI relative to thecontrol cells.

Any method may be used to introduce the gene into the host cell,including transfection with a retroviral vector, direct transfection(e.g., mediated by calcium phosphate or DEAE-dextran), andelectroporation. Preferably, a retroviral vector is used

The host cells should exhibit a readily observable phenotypic change asa result of enhanced production of the POI. Preferably, this responseshould be proportional to the level of production of the POI. Finally,the cells should not spontaneously manifest the desired phenotypicchange. For example, 3T3 cells form foci spontaneously. Among thepreferred cell lines for these methods are Rat-6 fibroblasts, C3H10T ½fibroblasts, and HL60. (HL60 is a human cell line that differentiates inresponse to PKC activation.) 3T3 cells may be used, but with thereservation stated above.

Generally speaking, it is preferable to maximize the ratio of productionby the “overproducing” cell line to production by the “native” line.This is facilitated by selecting a host cell line which produces littleor no POI, and introducing multiple gene copies and/or using high signalstrength promoters.

The Rat 6 embryo fibroblast cell line is a variant of the rat embryofibroblast cell line established by Freeman et. al., (1972) and isolatedby Hsiao et al., 1986. This cell line has an unusually flat morphology,even when maintained in culture at post-confluence for extended periodsof time, displays anchorage dependent growth and, thus far, has notundergone spontaneous transformation. It was also ideal for thesestudies since it has a very low level of endogenous PKC activity and alow level of high affinity receptors for phorbol esters.

According to these methods, one looks for is a increase in thephenotypic change exhibited by the cell which becomes greater withincreasing expression of the POI. This is a “graded cellular response,”and it is by this specialized response that inhibitors or activators ofthe POI can be distinguished from agents that act upon other cellmetabolites to effect a phenotypic change.

Thus, in a preferred embodiment, the cell lines are assayed for theirrelative levels of the POI, and their ability to grow inanchorage-independent systems (e.g., matrices such as soft agar ormethocel), to form small “foci” (areas of dense groups of cellsclustered together and growing on top of one another) in tissue culturedishes, to take up selected stains, or to bind an appropriately labeledantibody or other receptor for a cell surface epitope. In addition toexhibiting these growth control abnormalities, such cell lines will alsobe sensitive in their growth properties to chemical agents which arecapable of binding to, or modifying the biological effects of, the POI.

In selected embodiments, the method is particularly unique in that itcan be employed to search rapidly for EITHER activators OR inhibitors ofa given POI, depending upon the need. The term “activators,” as usedherein, includes both substances necessary for the POI to become activein the first place, and substance which merely accentuate its activity.The term “inhibitors” includes both substance which reduce the activityof the POI and these which nullify it altogether. When a POI has morethan one possible activity. The inhibitor or activator may modulate anyor all of its activities.

The use of this screening method to identify inhibitors or activators ofenzymes is of special interest. In certain preferred embodiments, themethod is used to identify inhibitors or activators of enzymes involvedin tumorigenesis and related phenomena, for example, protein kinase C,omithine decarboxylase, cyclic AMP-dependent protein kinase, the proteinkinase domains of the insulin and EGF receptors, and the enzyme productsof various cellular one genes such as the c-src or c-ras genes.

Protein kinase C (PKC) is a Ca and phospholipid-dependentserine/threonine protein kinase of fundamental importance in cellulargrowth control. PKC is activated endogenously by a wide variety ofgrowth factors, hormones, and neurotransmitters, and has been shown tobe a high affinity receptor for the phorbol ester tumor promoters aswell as other agents which possess tumor promoting activity (for reviewssee Nishizuka 1986; 1984; Ashendel, 1984). PKC has been shown tophosphorylate several intracellular protein substrates, including theepidermal growth factor (EGF) receptor (Hunter et al., 1984), pp60src(Gould et al., 1985), the insulin receptor (Bollag et al., 1986), p21ras (Jeng et al., 1987), and many others (Nishizuka, 1986). Severallaboratories have recently isolated cDNA clones encoding distinct formsof PKC, thus demonstrating that PKC is encoded by a multigene family(Ono et al., 1986, Knopf et al., 1986, Parker et al., 1986; Coussens etal., 1986; Makowske et al., 1986; Ohno et al., 1987; Housey et al.,1987). The multiple forms of PKC exhibit considerable tissue specificity(Knopf, et. al., 1986; Brandt et al., 1987; Ohno, et al., 1987; Housey,et. al., 1987) which suggests that there may be subtle differences inthe function(s) of each of the distinct forms. However, all of the cDNAclones which have been isolated thus far that encode distinct forms ofPKC share at least 65% overall deduced homology at the amino acid level,and transient expression experiments with some of these cDNA clones haveshown that they encode serine/threonine protein kinase activities whichbind to, or are activated by, the phorbol ester tumor promoters (Knopf,et. al., 1986, Ono, et. al., 1987).

With the exception of the brain, where its expression is very high,PKCbeta-1 is expressed at veer low levels in most tissues, and itsexpression is virtually undetectable in Rat 6 fibroblasts (see below).Thus, using this form will maximize the phenotypic differences observedbetween control cells and cells overexpressing the introduced form ofPKC. The PKCbeta-form is also of particular interest because within thePKC gene family its deduced carboxy terminal domain displays the highestoverall homology to the catalytic subunit of the cyclic AMP-dependentprotein kinase (PKAc) and the cyclic GMP-dependent protein kinase (PKG)(Housey et al., 1987). The latter observation suggests that PKAc, PKG,and the beta-1 form of PKC may share a common ancestral serine/threonineprotein kinase progenitor, and that the additional PKC genes may havebeen derived through evolutionary divergence from the beta-1 form.

Agents that interact with certain structural proteins, such as actin andmyosin, are also of interest. Mutations in the genes expressing theseproteins may be involved in tumorigenesis and metastasization. Suchinteractions can lead to changes in cell phenotype which can be assayedby this method.

In additional studies with other genes, most notably the c-H-rasoncogene, the catalytic subunit of the cyclic AMP-dependent proteinkinase, the c-myc oncogene, and certain cDNA clones encodingphorbol-ester inducible proteins, similar results have been obtained.Thus it is also clear that the method can be generalized to a videvariety of genes encoding proteins which are involved in cellular growthcontrol in numerous cell types.

One embodiment of a preferred protein inhibitor/activator drug screeningmethod of the invention can include the following steps:

1. Construction of an expression vector which is capable of expressingthe protein of interest in the selected host by inserting a geneencoding that protein into a transfer vector. The gene may be inserted3′ of a promoter already borne by the transfer vector, or a gene and apromoter may be inserted sequentially or simultaneously.

2. Introduction of the expression vector (a) into cells which producerecombinant retrovirus particles, or (b) directly into host cells whichwill be used for subsequent drug screening tests (the resulting cellsare called herein “test” cells). In parallel, the transfer vector (i.e.,the vector lacking the gene of interest and possibly a linked promoterbut otherwise identical to the expression vector) is preferably alsointroduced into the host cells. Cell lines derived from this latter casewill be used as negative controls in the subsequent drug screeningtests. Alternatively, the unmodified host cells may be used as controls.

If (a) was employed above, after an appropriate time (e.g., 48 hours),media containing recombinant virus particles is transferred onto hostcells so as to obtain test or control cells.

3. The test and control cells are transferred to selective growth mediumcontaining the appropriate drug which will only allow those cellsharboring the expression vector containing the selectable marker gene(as well as the gene or cDNA of experimental interest) to grow. After anappropriate selection time (usually 7-10 days), individual clones ofcells (derivative cell lines) are isolated and propagated separately.

4. Each independent cell line is tested for the level of production ofthe POI. By this method, a range of cell lines is generated whichoverproduce from a few fold to more that 100-fold levels of the POI. Inparallel, the control cell lines which contain only the transfer vectoralone (with the selectable marker gene) are also assayed for theirendogenous levels of the POI.

5. Each independent line is then tested for its growth capability insoft agar (or methocel, or any other similar matrix) of variouspercentages and containing different types of growth media until celllines are identified which possess the desired growth characteristics ascompared to the control cell lines.

6. Each cell line is also tested for its ability to form “foci”, orareas of dense cellular growth, in tissue culture plates using mediacontaining various percentages and types of serum (20%, 10%, 5% serum,fetal calf serum, calf serum, horse serum, etc.) and under variousconditions of growth (e.g. addition of other hormones, growth factors,or other growth supplements to the medium, temperature and humidityvariations, etc.). In these tests, the cells are maintained atpost-confluence for extended periods of time (from two to eight weeks)with media changes every three days or as required. Such growthparameters are varied until cell lines are identified which possess thedesired foci formation capacity relative to the control cell lines underthe identical conditions.

7. After a cell line possessing the required growth characteristics isidentified, the cells are grown under the conditions determined in (5)above with the growth medium supplemented with either crude or purifiedsubstances which may contain biologically active agents specific to thePOI. Thus, crude or purified substances possessing the latter propertiescan be rapidly identified by their ability to differentially alter thegrowth properties of the experimental cells (which overproduce the POI)relative to the control cells (which do not). This can be done rapidlyeven by simple observation with the naked eye, since the colonies whichgrow in soft agar after 2 weeks are easily seen even without staining,although they may be stained for easier detection.

Similarly, if the post-confluence foci formation assay is chosen, thefoci which result after approximately two weeks can be easily seen withthe naked eye, or these foci can also be stained. Results of the assayscan be rapidly determined by measuring the relative absorbance of thetest cells as compared to the control cells (at 500 nm, or anotherappropriate wavelength). In this fashion, thousands of compounds couldbe screened per month for their biological activity with very low laborand materials costs.

Furthermore, if antigen expression varies on the test cells expressinghigh levels of the POI relative to the control cells, a simpleEnzyme-Linked Immunoadsorption Assay (ELISA) could be performed and anantibody specific to the antigen.

While the assay may be performed with one control cell line and one testcell line, it is possible to use additional lines, tests lines withdiffering POI levels. Also additional sets of control/test lines,originating from other hosts, may be tested.

The system can also be used for identifying agents that bind to cellulartargets, such as membrane proteins, but without necessarily affecting oraltering the phenotype of the cell.

For example, the system may be used determine the ability of an agent tobind to a particular site on a membrane protein and thereby alter thelevel of surface expression thereof. Such an alteration in surfaceexpression may result from the agent blocking a site on the mutant thatcorresponds to an active site on the wild-type membrane protein and/orby blocking intracellular trafficking and/or processing of the mutantmembrane protein. Alternatively, an alteration in surface expression mayresult from the agent improving intracellular trafficking of the mutantmembrane protein.

As described above, there are a wide variety of formats known andavailable to those skilled in the art for appropriate binding assays.According to certain embodiments of the invention, one or more cellsexpressing a membrane POI may be provided in a suitable liquid mediumand exposed to one or more candidate compounds, while in otherembodiments the cells may be immobilized on a surface and then exposedto the candidate compound(s). Similarly, according to still otherembodiments of the invention, one or more candidate compounds may beimmobilized on a surface and exposed to a liquid medium containing oneor more cells that express a membrane protein of interest or thecandidate compound(s) may be included in a suitable liquid medium towhich one or more cells expressing a membrane protein of interest isadded.

Binding is often easier to detect in systems in which at least one ofthe candidate compound and the membrane POI is labeled (e.g., withfluorescence, radioactivity, an enzyme, an antibody, etch, includingcombinations thereof, as known to those skilled in the art). Afterexposing the candidate compound to the cell expressing a membraneprotein and washing off or otherwise removing unbound reagents, thepresence of the labeled moiety (i.e., bound to the unlabelled componentof the test system) is measured.

Methods for performing various binding assays are known in the art,including but not limited to the assay systems such as those describedin PCT Application US98/18368. Various references provide generaldescriptions of various formats for protein binding assays, includingcompetitive binding assays and direct binding assays, (see e.g., Stitesand Terr, Basic and Clinical Immunology, 7th ed. (1991); Maggio, EnzymeImmunoassay, CRC Press, Boca Raton, Fla. (1980); and Tijssen, Practiceand Theory of Enzyme Immunoassays, in Laboratory Techniques inBiochemistry and Molecular Biology, Elsevier Science Publishers, B.V.Amsterdam, (1985)).

Thus, according to certain embodiments, immunoassays are provided inwhich one or more cells expressing a membrane protein of interest aregenerally bound to a suitable solid support and combined with acandidate agent, and observing changes in the level of surfaceexpression of the membrane POI. In these embodiments, one or more of theassay components is attached to a solid surface.

In some embodiments, an assay system may used (as known in the art) todetect the change in the surface expression of the membrane protein dueto the binding of the candidate agent. For example, if the membraneprotein of interest is a membrane ion channel, a patch clamp assay maybe employed to detect a change in the flux of ions across the membrane,thus evidencing an increase in the level of surface expression of theion channel.

In alternative embodiments, an indirect immunoassay system is used inwhich the membrane protein on the surface of the cell(s) is detected bythe addition of one or more antibodies directed against an extracellularepitope of the membrane protein, as known in the art.

When using a solid support in embodiments of methods according to theinvention, virtually any solid surface is suitable, as long as thesurface material is compatible with the assay reagents and it ispossible to attach the component to the surface without unduly alteringthe reactivity of the assay components. Those of skill in the artrecognize that some components exhibit reduced activity in solid phaseassays, but this is generally acceptable, as long as the activity issufficient to be detected and/or quantified. Suitable solid supportsinclude, but are not limited, to any solid surface such as glass beads,planar glasses, controlled pore glasses, plastic porous plastic metals,or resins to which a material or cell may be adhered, etc.). Those ofsill in the art recognize that in some embodiments, the solid supportsused in the methods of the invention may be derivatized with functionalgroups (e.g., hydroxyls, amines, carboxyls, esters, and sulfhydryls) toprovide reactive sites for the attachment of linkers or the directattachment of the candidate agent or other assay component.

Adhesion of an assay component to a solid support can be direct (i.e.the component directly contacts the solid surface) or indirect (i.e. anagent and/or component (e.g. an antibody) is/are bound to a support, andthe other assay component(s) binds to this agent or component ratherthan to the solid support). In some embodiments, the agent or componentis covalently immobilized (egg, utilizing single reactive thiol groupsof cysteine for anchoring proteinaceous components (see e.g., Bioconjug.Chem., 4:528-536 (1993)), or non-covalently, but specifically (e.g., viaimmobilized antibodies or other specific binding proteins (see e.g.,Adv. Mater., 3:388-391 (1991); Anal. Chem., 67:83-87 (1995))), thebiotin/streptavidin system (see e.g., Biophys. Biochem. Res. Commun.,230:76-80 (1997)), or metal-chelating Langmuir-Blodgett films (see e.g.,Langmuir 11:4048-4055 (1995); Angew. Chem. Int. Ed. Engl., 35:317-320(1996); Proc. Natl. Acad. Sci. USA 93:4937-4941 (1996); and J. Struct.Biol., 113:117-123 (1994)), and metal-chelating self-assembledmonolayers (see e.g., Anal. Chem., 68:490-497 (1996)), for binding ofpolyhistidine fusion proteins.

In some embodiments, standard direct or indirect ELISA, IFA, or RIAmethods as generally known in the art are used to detect the binding ofa candidate agent to a membrane POI. In some embodiments, an increase inthe level of surface expression of the membrane protein is detected in asample; while in other embodiments, a decrease in the level of surfaceexpression is detected. Thus, it is clear that embodiments of methodsaccording to the invention are adaptable to the detection,identification, and characterization of multiple elements.

Accordingly, in some particularly preferred embodiments of the methodsof the invention, a sandwich ELISA (enzyme-linked immunosorbent assay)with a monoclonal or polyclonal antibody for capture (“a captureantibody”) and a secondary antibody (“a reporter antibody”) fordetection of bound antibody-antigen complex may be used.

In some preferred ELISA embodiments, alkaline phosphatase conjugates areused, while in still other preferred embodiments, horseradish peroxidaseconjugates are used. In addition, avidin/biotin systems may also beused, particularly for assay systems in which increased signal aredesired. Suitable enzymes for use in preferred embodiments include, butare not limited to, peroxidases, luciferases, alkaline phosphatases,glucose oxidases, beta-galactosidases and mixtures of two or morethereof.

In addition to the assay systems in which a solid support is utilized,the invention pro-ides embodiments of methods in which the assaycomponents remain suspended in solution.

Any change, such as an increase or decrease, in the level of binding inthe presence of the candidate agent relative to control indicates thatthe candidate agent alters the level of surface expression of the firstmutant form of the membrane protein.

The determination of the level of surface expression of the integralmembrane protein of interest may be performed using any of the methodsand techniques known and available to those skilled in the art.Preferably, the level of binding is determined by fluorescence,luminescence, radioactivity, absorbance or a combination of two or moreof these.

According to certain embodiments of the invention, the extracellularepitope to which the antibody binds on the membrane protein ispreferably the same as a wild-type epitope, i.e. an extracellularepitope found on the naturally-occurring form(s) of the membrane proteinof interest. Without wishing to be bound to any theory of operability orthe line, such an arrangement may have the potential to reduce errorsarising from differences in protein structure, for example by a changein one or more of the functional properties of the protein.

According to certain embodiments of the invention, the extracellularepitope may also contain a tag. Suitable tags are known and available tothose skilled in the art. A particularly preferred tag for use inselected methods of the invention is a hemagglutinin (HA) tag. The tagmay be inserted in an extracellular domain of the POI or may replace aportion of an extracellular domain thereof.

A method of identifying an agent that alters the level of surfaceexpression or binds to an extracellular epitope of a membrane protein ina mammalian cell according to disclosed embodiments can includepreparing a first medium containing mammalian cells that express themembrane protein, adding to the first medium containing mammalian cellsan effective amount of a candidate agent, incubating the cells in thepresence of the candidate agent for a sufficient period of time in asystem according to embodiments, adding to the first medium containingmammalian cells an effective amount of at least one antibody which bindsto at least one extracellular epitope of the membrane protein anddetermining the level of binding of the at least one antibody to theextracellular epitope following incubation with the candidate agent,wherein a change in the level of binding relative to control indicatesthat the candidate agent alters the level of surface expression of themembrane protein or binds to the extracellular epitope of the membraneprotein.

A method for detecting the presence of a protein or gene of interest ina sample, according to one embodiment can include (a) placing a samplein a system according embodiments, (b) contacting the sample with acompound which selectively hybridizes to the gene of interest or bindsto the protein and (c) determining whether the compound hybridizes tothe gene of interest or binds to the protein in the sample.

A method for identifying a compound which binds to or modulates theactivity of a protein according to one embodiment can include (a)immobilizing a cell expressing the protein in a system accordingembodiments, (b) contacting the cell with a test compound and (c)determining whether the protein binds to the test compound ordetermining the effect of the test compound on the activity of theprotein.

A method of identifying a nucleic acid molecule associated with adisorder or identifying a subject having a disorder or at risk fordeveloping a disorder according to embodiments of the invention caninclude (a) placing a sample containing nucleic acid molecules from asubject with or at risk of developing a disorder in a system accordingembodiments, (b) contacting the sample with a hybridization probe thatcontains a nucleic acid sequence indicative of the disorder or risk fordeveloping the disorder and (c) detecting the presence of a nucleic acidmolecule in the sample that hybridizes to the probe, thereby identifyinga nucleic acid molecule associated with a disorder or the subject havingthe disorder or at risk for developing the disorder.

In accordance with another embodiment, a pharmaceutical composition caninclude a therapeutically effective amount of an agent identified ordescribed herein and a pharmaceutically effective carrier.

In accordance with another embodiment, a method can include adding aneffective amount of at least one primary antibody and an effectiveamount of at least one secondary antibody, wherein the primary antibodybinds to an extracellular epitope of a membrane protein and thesecondary antibody binds to the first antibody. In accordance withanother embodiment, a level of binding can be measured by fluorescence,luminescence, radioactivity, absorbance or a combination of two or morethereof.

In accordance with another embodiment, an integral membrane protein canbe a membrane ion channel. In accordance with another embodiment, amembrane ion channel can be a sodium channel, a potassium channel, acalcium channel or a chloride channel.

In accordance with another embodiment, a primary or secondary antibodycan be coupled to an enzyme. In accordance with another embodiment, anenzyme can be selected from the group including or consisting ofperoxidases, luciferases, alkaline phosphatases, glucose oxidases,beta-galactosidases, or the like and mixtures of two or more thereof.

Other applications in the tissue regeneration and engineering, clinical,and research industries include, but are not limited to, tissueengineering arteries and veins for cardiovascular repair or replacementand training ex vivo veins or arteries prior to implantation or applyinga treatment to the specimen prior to implantation. For example,embodiments of the system can simulate complex coronary hemodynamics forgrowing tissue engineered or regenerated arteries and for trainingsaphenous veins or defrosting cryogenic arteries in preparation for theharsh and dynamic coronary environment or other peripheral arterialdisease regions. Another example may include treating an artery or veinwith gene, RNAi, or other biomolecular therapy in conjunction withhemodynamic simulation prior to therapeutic intervention. Embodiments ofthe system can provide accurate and precise control of physiologicparameters for applications in the tissue regeneration and engineeringindustries. For example, to grow vascular grafts seeded with stem cells,hemodynamic stimuli ranging from coronary to peripheral arteries, aswell as biochemical stimuli, such as growth factors, can be applied tocondition the stem cells to differentiate to vascular cells that arepreferable functional.

The surfaces of synthetic vascular prostheses are capable of provokingplatelet activation and blood coagulation, generating clots that canrapidly occlude the engrafted prosthetic. Thus, the field of syntheticvascular grafts has developed at a cautious pace, and efforts to ensuretheir safety have included the testing of different graft materials andthe inclusion of anti-thrombogenic materials in the pre-treatment usedto seal the interstices of the graft to prevent blood loss from thevessel. (Sausage, L. R., in Haimovici et al., eds., Haimovici's VascularSurgery, 4th ed., 1996). Today, only polyethylene terephthalate (DACRON)and polytetrafluoroethylene (TEFLON) are approved by the Federal DrugAdministration for this use.

Even so, autologous grafts still are considered superior to syntheticones because their endothelial linings, which secrete a number ofnatural anti-thrombotic substances, provide a far better flow surfacethan the material used for today's synthetic prostheses. Unfortunately,only a limited number of the body's blood vessels provide tissuesuitable for use in autologous vascular transplants, and improvements inthe field of synthetic prostheses would prove a boon to many patients,especially those requiring multiple heart bypasses.

Another limitation of synthetic vascular prostheses currently approvedfor use is that the caliber, i.e., inner diameter, of grafts deemed asacceptable must be at least 6 mm. It is believed, in fact, that nosatisfactory synthetic prosthesis having a caliber below 6 mm existstoday (e.g., Sauvage, 1996). Thus, the need for smaller caliber graftsremains unfulfilled, even though numerous patients require repeatcoronary bypass, or have peripheral arterial occlusions below the kneeor in the cerebrovascular tree, which would use small caliber syntheticgrafts if these were available.

In recent years, a number of investigators have reported the occasionalappearance of patches of endothelial cells growing on the walls ofsynthetic vascular grafts (e.g., Wu et al., J. Vasc. Surg. 21:862-867,1995; Scott et al., J. Vasc. Surg. 19:585-593, 1994; Shi et al., J.Vasc. Surg. 25:736-742, 1997). Several studies have suggested that thisgraft surface endothelialization originates primarily from transmuralmicrovessels, i.e., tiny blood vessels that infiltrate the graft wall,and that originate themselves from pre-existing blood vessels (e.g.,Cloves et al., Am. J Pathol. 123:220-230, 1986; Wu et al., Ann. Vasc.Surg. 10:11-15, 1996). However, other studies have indicated that atleast some of the endothelialization observed in internal segments ofsynthetic vascular grafts appears to originate from blood-borne cellsthat became attached to the vessel walls (Scott et al., J. Vasc. Surg.19:585-593, 1994; Shi et al., J. Vasc. Surg-20:546-555, 1994; Wu et al.J. Vasc. Surg. 21:862-867, 1995; Shi et al. J Vasc. Surg. 25:736-42,1997; Frazier et al. Tex. Heart Inst. J 20:78-82, 1993; Hammond et al.,Blood 88 (suppl. 1):511a (abstract, 1996)). This phenomenon is called“fallout endothelialization.” More specifically, it has been proposedthat the circulating cells that give rise to endothelial coatings ofvascular prostheses may arise from the bone marrow (Hammond et al.1996).

Indeed, circulating endothelial cells have been observed by manyinvestigators (Asahara et al., Science 275:965-967, 1997; Percivalle etal. J. Clin. Invest. 92:663-670, 1993; George et al. ThrombosisHaemostasis 67:147-153, 1992). The latter two of these provide evidencethat circulating endothelial cells originate from the walls of bloodvessels (George et al., 1992; Percivalle et al., 1993), and the study ofAsahara et al. (1997) provides evidence for a circulating endothelialprogenitor cell that expresses CD34, an antigen also associatedhematopoietic progenitor cells, and that can participate in angiogenesisin ischemic tissues. Whatever their source, graft recipients clearlywould benefit from the development of treatments promoting thedeposition and outgrowth of circulating endothelial cells on the innerwalls of synthetic vascular grafts.

In view of the superior anti-thrombotic properties of endothelial flowsurfaces, various experimental approaches have been devised forincreasing the rate of endothelialization of synthetic grafts. Theseinclude seeding prior to implant with autogenous endothelium, culturedendothelium or bone marrow cells (Herring et al. Surgery 84:498-504,1978; Anderson et al., Surgery 101-577-586, 1987; Kadletz et al., J.Thorac. Cardiovasc. Surg. 104:736-742, 1992; Mazzucotelli et al., ArtifOrgans 17:787-790, 1993; Noishiki et al., Artif. Organs 19:17-26, 1995;Noishiki et al., Nat. Med. 2:90-93, 1996; Onuki et al. Ann. Vasc. Surg.11:141-148, 1997). None of these, however, have been able to replicatethe in vivo hemodynamic environment necessary for completeendothelialization with a confluent monolayer of cells that can beachieved with embodiments of the system according to the invention.

One application of exemplary embodiments of systems and methodsdescribed herein is combination (or hybrid) medical devices and celltherapy. A hybrid or combination vascular graft is one exemplary medicaldevice. A hybrid vascular graft is made up of both synthetic materialand living cells. Embodiments of a hybrid or combination vascular graftwill now be described. Embodiments of a hybrid vascular graft can bedeveloped using exemplary embodiments of systems and methods describedherein (e.g. FIGS. 1-48), however, an embodiment of the hybrid vasculargraft is not intended to be so limited thereby.

Fin embodiment of a hybrid vascular graft can be a synthetic vasculargraft (e.g., silicon) combined with living cells (e.g., a biomaterial)that can reduce or eliminate the need for the costly dependence ondrugs, reduce subsequent surgeries and more accurately reflect humanbiology. For example, the hybrid graft embodiment can recapitulatenative function and/or the living cells can be functional endothelialcells (e.g., evidenced by cell characteristics, expression profiles ormetabolism). The hybrid graft embodiment can replicate the originalphysiologic function of living arteries and veins with vascular cells.Further, the hybrid graft embodiment can be used for the difficult orpreviously impossible small diameter synthetic grafts (e.g., 6 mm orless, 4 mm or less). The hybrid graft embodiment can use endothelialcells or other cells (e.g., stem cells) that differentiate into inendothelial cells that are attached to a synthetic graft. Once cells(e.g., endothelial cells) are attached (e.g., as conventionally known)to the synthetic graft a functional coating (e.g., a confluentmonolayer) of cells is grown on the hybrid grafts using disclosedsystems and methods (e.g., FIGS. 36-39).

According to one embodiment of a hybrid vascular graft (combinationsynthetic vascular graft) or combination medical device, it can be usedfor synthetic vascular grafts for selected uses, including 1)hemodialysis access vascular graft, 2) femoral artery graft and/or 3)coronary bypass graft. Additional exemplary uses of embodiments of ahybrid graft can include coronary replacements, repair of obstructivedisease, aneurysm repair, trauma repair, cardiovascular diseasetreatment or the like.

Arterial diseases include Peripheral Arterial Disease (PAD), which isthe build up of fat on the artery wall and narrowing of the arterystructure limiting blood supply and atherosclerosis. PAD can occur inlocations, such as carotid artery, renal artery, iliac artery, femoralartery, popliteal artery, or tibial artery. Atherosclerosis is a chronicdisease in which thickening, hardening, and loss of elasticity of thearterial walls result in impaired blood flow. In addition, vascularfailures can cause diseases including angina, high blood pressure, highcholesterol, heart attack, stroke, and arrhythmia.

Treatment of such diseases can include bypass or graft surgery. Anexemplary double bypass graft can use one bypass to connect the internalmammary artery to a branch of the left coronary artery and the otherbypass to connect the aorta to the right coronary artery. A major modeof treatment for cardiovascular diseases using bypass or graft surgeryis via synthetic vascular grafts.

However, disadvantages of prosthetics or synthetic vascular graftsinclude mechanical disadvantages, such as poor compliance (e.g., rigid),size mismatch and viscoelasticity, and biocompatibility disadvantages.Biocompatibility complications include intimal hyperplasia atanastamoses, thrombosis, restenosis (rapid uptake), infection, bacteriacolonization, dilatation or rupture. Vascular grafts can also failbecause of compliance mismatch, such as within the bulk material, withinthe sutured attachment to the existing vessel or at the anastamosis.

A hemodialysis access graft though an arterio-venous (AV) shunt is alooped graft between an artery and a vein (e.g., in the body). Forexample, the AV shunt can be located in the upper arm, middle arm, lowerarm or combinations thereof. The blood can be transferred to a dialysismachine from the portion of the AV shunt connected to the artery andreturned from the dialysis machine to the portion of the AV shuntconnected to the vain.

Stents elicit negative reactions from the body since the material isnon-living or non-biological, and thus subsequently fail because ofre-closure of a treated blood vessel caused by growth of smooth musclecells, stent thrombosis, and structural/mechanical failure of the graftor the like.

In contrast, embodiments of hybrid grafts can consider the biophysicalenvironment the graft will be in, such as, for example, thecardiovascular system, including simulation of in vivo hemodynamics(e.g., concurrent wall shear stress, stretch, and pressure). Embodimentsof the hybrid vascular graft can be processed in vitro to grow or trainendothelial cells on the vascular graft surface (e.g., tubularstructure) that can function as if it was in a desired in vivoenvironment. Related art technologies cannot grow cells on a vasculargraft let alone functional endothelial cells because stretch devicesproduce only a biaxial or heterogeneous strain field without appliedflow, and flow devices produce only a flow field without stretch.

Embodiments of hybrid vascular grafts can utilize/train with regard to aphysical nature of a graft or disease, a dynamic environment of a graftand/or the dynamic nature of disease. Further, embodiments of hybridvascular grafts can be developed at a size greater than 6 mm, but alsoat a size 6 mm or less, 5 mm or less, 4 mm or less or the like and havesignificantly reduced risks or clogging or thrombosis. Such riskreduction is achieved by training the endothelial cells or stem cellsthat ultimately differentiate into endothelial cells that yieldsfunctional endothelial cells that line the hybrid graft.

One embodiment of a hybrid graft can be developed using a syntheticvascular graft provided with stem cells (as is known in the art, e.g.,vascular cell origin from hemangioblast) and exposed to controlledhemodynamics resulting in an exemplary graft with functional vascularendothelial cells. Such an exemplary embodiment of a hybrid vasculargraft with functional vascular endothelial cells can be used asdescribed above. According to another embodiment, stem cells can beextracted from the patient intended to receive the embodiment of ahybrid vascular graft (a combination synthetic graft).

In one embodiment for preparing a hybrid vascular graft, a plurality ofcells is affixed to a surface of a synthetic graft. A binding material(e.g., adhesion proteins, fibronectin) can be used to coat a surface ofthe synthetic graft to affix the plurality of cells. In anotherembodiment for preparing a hybrid vascular graft, etching of thesynthetic graft can improve surface adhesion of proteins and cells thatcan reduce or remove the necessity of a binding material. Etching withplasma treatments can include oxygen plasma, glow-discharge plasmas oramide and amine containing plasmas. Further, for polytetrafluorethylene(PTFE) or ePTFE, ammonia and oxygen plasmas can be used and fluorine canbe replaced with amines and nitrogen groups to help facilitate adhesionof proteins and cells (e.g., EC).

Additional exemplary embodiments of methods for processing biomaterials,non-biomaterials or combinations thereof (e.g., hybrid vascular grafts)will now be described.

An exemplary method embodiment of preparing a biomaterial intended forimplantation into a mammal in need thereof can include placing thebiomaterial in a system according embodiments of the invention for asufficient time prior to implantation of the biomaterial into themammal. An exemplary method embodiment of promoting engraftment of abiomaterial following implantation into a mammal's body can includeplacing the biomaterial in a system according to disclosed embodimentsprior to implantation of the biomaterial into the mammal's body.

As shown in FIG. 49, an exemplary method of using systems disclosedherein (e.g., systems 1, 1101) for treating or processing a biomaterialwill now be described, As shown in FIG. 49, selected biomaterials suchas cells (e.g., endothelial cells, stem cells) can be combined with anon-biomaterial (e.g., a synthetic graft) (block 4905). The combinationcan then be placed in a simulator of a selected class of dynamicconditions (e.g., selected system embodiment 1, 1101) (block 4910). Thecombination is then exposed to a selected or prescribed dynamiccondition or series of conditions (e.g., hemodynamic conditions of anabdominal aorta) (block 4915). The combination (e.g., a hybrid syntheticgraft) can then be continuously monitored or periodically monitored fordesired results (e.g., generation of a confluent functional monolayer ofendothelial cells) or for a selected period of time (block 4920).Optionally, the controlled dynamic conditions can be repeated ormodified (e.g., “dial-up”) according to feedback (e.g., FB_(j)(t)) fromthe monitored combination or its environment or desired results (block4925). When the time periods have elapsed or results have been obtained,the combination can be extracted from the selected dynamic conditionclass simulator (block 4830). The modified combination can then beimplanted in a mammal. The method embodiment of FIG. 49 can be performedon biomaterials alone.

An exemplary method embodiment of promoting endothelialization of avascular graft can include (a) immobilizing a plurality, of endothelialcells on at least one surface of a vascular graft and (b) placing thevascular graft in a system according to disclosed embodiments underconditions effective to promote the endothelial cells to form aconfluent monolayer on the surface of the vascular graft.

An exemplary method embodiment of coating a vascular graft with aconfluent monolayer of endothelia cells can include (a) immobilizing aplurality of endothelial cells on at least one surface of a vasculargraft; and (b) placing the vascular graft in a system accordingdisclosed embodiments under conditions effective to promote theendothelial cells to form a confluent monolayer on the surface of thevascular graft. An exemplary method embodiment of coating a vasculargraft with a confluent monolayer of endothelial cells can include (a)immobilizing a plurality of multipotent stem cells on at least onesurface of a vascular graft, (b) placing the vascular graft in a systemaccording to disclosed embodiments under conditions effective to promotethe stem cells to form confluent monolayer on the surface of thevascular graft and (c) placing the vascular graft in an environment thatpromotes the stem cells to differentiate into endothelial cells.

An exemplary method embodiment of promoting endothelialization of avascular graft can include (a) immobilizing a plurality of multipotentstem cells on at least one surface of a vascular graft; and (b) placingthe vascular graft in a system according to disclosed embodiments underconditions effective to promote the stem cells to form a confluentmonolayer on the surface of the vascular graft or to differentiate intoendothelial cells on the surface of the vascular graft.

An exemplary method embodiment for the generation of tissue can include(a) immobilizing a plurality of cells in at least one surface of amatrix, the matrix including a suitable biomedical material; and (b)placing the matrix in a system according to disclosed embodiments underconditions effective to promote the cells to grow on the surface of thematrix. An exemplary method embodiment of storing an organ prior totransplantation into a patient in need thereof; can include placing theorgan in a system according to disclosed embodiments under conditions inwhich the organ remains substantially unchanged or viable for anextended period of time.

In accordance with another embodiment, a coating including at least onecell is applied to at least a portion of at least one surface of thebiomaterial (e.g., vascular graft, matrix or the like) prior toplacement in the system. In accordance with another embodiment, the cellis selected from embryonic stem cells, adult stem cells, mesenchymalstem cells, endothelial cells, smooth muscle cells, osteocytes, orosteoblasts. In accordance with another embodiment, the coating caninclude an affixing substance selected from fibronectin, fibrin glue,combinations of fibrinogen and thrombin, collagen, basement membrane, oralginate, and mixtures of two or more thereof.

In accordance with another embodiment, the coating further can includeat least one supplement selected from an analgesic, an anesthetic, anantimicrobial compound, an antibody, an anticoagulant, anantifibrinolytic agent, an anti-inflammatory compound, an antiparasiticagent, an antiviral compound, a cytokine, a cytotoxin or cellproliferation inhibiting compound, a chemotherapeutic drug, a growthfactor, an osteogenic or cartilage inducing compound, a hormone, aninterferon, a lipid, an oligonucleotide, a polysaccharide, a proteaseinhibitor, a proteoglycan, a polypeptide, a steroid, a vasoconstrictor,a vasodilator, a vitamin, or a mineral, and mixtures of any two or morethereof.

In accordance with another embodiment, a supplemented coating is appliedto the biomaterial in an amount effective to promote cell migration,cell proliferation and/or cell differentiation in a cell-containingenvironment. In accordance with another embodiment a supplementedcoating is applied to the biomaterial in an amount effective to promoteendothelialization of the biomaterial in an endothelial cell-containingenvironment, where such endothelialization can cause a confluent layerof cells to form on the surface of the biomaterial when the biomaterialis placed into the endothelial cell-containing environment. Inaccordance with another embodiment, a supplemented coating is applied tothe biomaterial in an amount effective for the prophylaxis or treatmentof infection in a patient when the biomaterial is placed into a patient.

In accordance with another embodiment, a biomaterial can include orcombine with an orthopedic device, a urinary catheter, an intravascularcatheter, a suture, a vascular prosthesis, an intraocular lens, acontact lens, a heart valve, a shoulder replacement device, an elbowreplacement device, a hip replacement device, a knee replacement device,an artificial heart, a fixation plate, a dental implant, a nasalimplant, a breast implant, a testicular implant, a sponge, a film or abag. In accordance with another embodiment, a biomaterial can beprepared according to such exemplary method embodiments.

In accordance with another embodiment, the biomaterial can be combinedwith a synthetic vascular graft or prosthesis. In accordance withanother embodiment, the biomaterial intended for implantation into amammal includes a synthetic vascular graft or hybrid vascular graft.

In accordance with another embodiment, the biomaterial intended forimplantation into a mammal can be used for a hybrid hemodialysis accessgraft, a hybrid femoral artery graft or a hybrid coronary bypassvascular graft. In accordance with another embodiment, a hybrid vasculargraft is one of at least 8 mm, less than 8 mm, in less than 6 mm, lessthan 5 mm, less than 4 mm less than 3 mm less than 2 mm less than 1 mm,or less than 0.5 mm in diameter.

Embodiments of the system may also be used to differentiateundifferentiated cells, such as, for example, adult stem cells orprogenitor cells, toward a particular differentiated state such as, forexample, an adult stem cell to an endothelial cell or a smooth musclecell. The technology can also be used to train or condition cells ortissue such as saphenous vein or tissue engineered artery or vein.Organs or tissues can also be used in embodiments of the system toprovide the correct physiologic simulation for various applications suchas research, development, organ transport, or the construction of a morein vivo like system and the like. Embodiments of the system can also beused to maintain, grow, or enhance the growth of various organs, cells,and tissues such as liver, kidney, heart, bone, or synovial tissue.

The most promising source of organs and tissues for transplantation liesin the development of stem cell technology. Theoretically, stem cellscan undergo self-renewing cell division to give rise to phenotypicallyand genotypically identical daughters for an indefinite time andultimately can differentiate into at least one final cell type. Bygenerating tissues or organs from a patient's own stem cells, or bygenetically altering heterologous cells so that the recipient immunesystem does not recognize them as foreign, transplant tissues can begenerated to provide the advantages associated with xenotransplantationwithout the associated risk of infection or tissue rejection.

Stem cells also provide promise for improving the results of genetherapy. A patient's own stem cells could be genetically altered invitro, then reintroduced in vivo to produce a desired gene product.These genetically altered stem cells would have the potential to beinduced to differentiate to form a multitude of cell types forimplantation at specific sites in the body, or for systemic application.Alternately, heterologous stem cells could be genetically altered toexpress the recipient's major histocompatibility complex (MHC) antigen,or no MHC, to allow transplant of those cells from donor to recipientwithout the associated risk of rejection.

Stem cells are defined as cells that have extensive, perhaps indefinite,proliferation potential di-t differentiate into several cell lineages,and that can repopulate tissues upon transplantation. The quintessentialstem cell is the embryonal stem (ES) cell, as it has unlimitedself-renewal and multipotent differentiation potential. These cells arederived from the inner cell mass of the blastocyst, or can be derivedfrom the primordial germ cells from a post-implantation embryo(embryonal germ cells or EG cells). ES and EG cells have been derivedfrom mouse, and more recently also from non-human primates and humans.When introduced into mouse blastocysts or blastocysts of other animals,ES cells can contribute to all tissues of the mouse (animal). Whentransplanted in post-natal animals, ES and EG cells generate teratomas,which again demonstrates their multipotency. ES (and EG) cells can beidentified by positive staining with the antibodies SSEA1 and SSEA4.

At the molecular level, ES and EG cells express a number oftranscription factors highly specific for these undifferentiated cells.These include oct-4 and Rex-1. Also found are the LIF-R and thetranscription factors sox-2 and Rox-1, even though the latter two arealso expressed in non-ES cells. Oct-4 is a transcription factorexpressed in the pregastrulation embryo, early cleavage stage embryo,cells of the inner cell mass of the blastocyst, and in embryoniccarcinoma (EC) cells. Oct-4 is down-regulated when cells are induced todifferentiate in vitro and in the adult animal oct-4 is only found ingerm cells. Several studies have shown that oct-4 is required formaintaining the undifferentiated phenotype of ES cells, and plays amajor role in determining early steps in embryogenesis anddifferentiation. oct-4, in combination with Rox-1, causestranscriptional activation of the Zn-finger protein Rex-1, and is alsorequired for maintaining ES in an undifferentiated state. Likewise,sox-2, is needed together with oct-4 to retain the undifferentiatedstate of ES/EC and to maintain murine (but not human) ES cells. Human ormurine primordial germ cells require presence of LIF. Another hallmarkof ES cells is presence of telomerase, which provides these cells withan unlimited self-renewal potential in vitro.

Stem cells have been identified in most organ tissues. The bestcharacterized is the hematopoietic stem cell. This is a mesoderm-derivedcell that has been purified based on cell surface markers and functionalcharacteristics. The hematopoietic stem cell, isolated from bone marrow,blood, cord blood, fetal liver and yolk sac, is the progenitor cell tintreinitiates hematopoiesis for the life of a recipient and generatesmultiple hematopoietic lineages (see Fei, R., et al., U.S. Pat. No.5,635,387; McGlave, et al., U.S. Pat. No. 5,460,964; Simmons, P., etal., U.S. Pat. No. 5,677,136; Tsukamoto, et al., U.S. Pat. No.5,750,397; Schwartz, et al., U.S. Pat. No. 759,793; DiGuisto, et al.,U.S. Pat. No. 5,681,599; Tsukamoto, et al., U.S. Pat. No. 5,716,827;Hill, B., et al., Exp. Hematol. (1996) 24 (8): 936 943). Whentransplanted into lethally irradiated animals or humans, hematopoieticstem cells can repopulate the erythroid, neutrophil-macrophage,megakaryocyte and lymphoid hemopoietic cell pool. In vitro, hemopoieticstem cells can be induced to undergo at least some self-renewing celldivisions and can be induced to differentiate to the same lineages as isseen in vivo. Therefore, dais cell fulfills the criteria of a stem cell.Stem cells which differentiate only to form cells of hematopoieticlineage, however, are unable to provide a source of cells for repair ofother damaged tissues, for example, heart or lung tissue damaged byhigh-dose chemotherapeutic agents.

A second stem cell that has been studied extensively is the neural stemcell (Gage F H: Science 287:1433 1438, 2000; Svendsen C N et al, BrainPath 9:499 513, 1999; Okabe S et al, Mech Dev 59:89 102, 1996). Neuralstem cells were initially identified in the subventricular zone and theolfactory bulb of fetal brain. Until recently, it vas believed that theadult brain no longer contained cells with stem cell potential. However,several studies in rodents, and more recently also non-human primatesand humans, have shown that stem cells continue to be present in adultbrain. These stem cells can proliferate in vivo and continuouslyregenerate at least some neuronal cells in vivo. When cultured ex vivo,neural stem cells can be induced to proliferate, as well as todifferentiate into different types of neurons and glial cells. Widentransplanted into the brain, neural stem cells can engraft and generateneural cells and glial cells. Therefore, this cell too fulfills thedefinition of a stem cell.

Mesenchymal stem cells (MSC), originally derived from the embryonalmesoderm and isolated from adult bone marrow, can differentiate to formmuscle, bone, cartilage, fat, marrow stroma, and tendon. Duringembryogenesis, the mesoderm develops into limb-bud mesoderm, tissue thatgenerates bone, cartilage, fat, skeletal muscle and possiblyendothelium. Mesoderm also differentiates to visceral mesoderm, whichcan give rise to cardiac muscle, smooth muscle, or blood islandsconsisting of endothelium and hematopoietic progenitor cells. Primitivemesodermal or mesenchymal stem cells, therefore, could provide a sourcefor a number of cell and tissue types. A third tissue specific cell thathas been named a stem cell is the mesenchymal stem cell, initiallydescribed by Fridenshtein (Fridenshtein, Arkh. Patol., 44.3 11, 1982). Anumber of mesenchymal stem cells have been isolated (see, for example,Caplan, A., et al., U.S. Pat. No. 5,486,359; Young, H., et al., U.S.Pat. No. 5,827,735; Caplan, A., et al., U.S. Pat. No. 5,811,094; Bruder,S., et al., U.S. Pat. No. 5,736,396; Caplan, A., et al., U.S. Pat. No.5,837,539; Masinovsly, B., U.S. Pat. No. 5,837,670; Pittenger, M., U.S.Pat. No. 5,827,740; Jaiswal, N., et al., J. Cell Biochem. (1997) 64(2):295 312; Cassiede P., et al., J. Bone Miner. Res. (1996) 11(9): 12641273; Johnstone, B., et al., (1998) 238(1): 265 279; Yoo, et al., J.Bone joint Sure. Am. (1998) 80(12). 1745 1757; Gronthos, S., Blood(1994) 84(12): 416-44173; Makino, S., et al., J. Clin. Invest. (1999)103(5): 697 705).

Other stem cells have been identified, including gastrointestinal stemcells, epidermal stem cells, and hepatic stem cells, also termed ovalcells (Potten C, Philos Trans R Soc Lond B Biol Sci 353:821 30, 1998;Watt F, Philos. Trans R Soc Lond B Biol Sci 353:831, 1997; Alison M etal, Hepatol 29:678 83, 1998).

Compared with ES cells, tissue specific stem cells have lessself-renewal ability and, although they differentiate into multiplelineages, they are not multipotent. No studies have addressed whethertissue specific cells express markers described above of ES cells. Inaddition, the degree of telomerase activity in tissue specific stemcells has not been fully explored, in part because large numbers ofhighly enriched populations of these cells are difficult to obtain.

Until recently, it was thought that organ specific stem cells could onlydifferentiate into cells of the same tissue. A number of recentpublications have suggested that adult organ specific stem cells may becapable of differentiating into cells of different tissues. A number ofstudies have shown that cells transplanted at the time of a bone marrowtransplant can differentiate into skeletal muscle (Ferrari Science279:528 30, 1998; Gussoni Nature 401:390 4, 1999). This could beconsidered within the realm of possible differentiation potential ofmesenchynmal cells that are present in marrow. Jackson published thatmuscle satellite cells can differentiate into hemopoietic cells, again aswitch in phenotype within the splanchnic mesoderm Jackson PNAS USA96:14482 6, 1999). Other studies have shown that stem cells from oneembryonal layer (for instance splanchnic mesoderm) can differentiateinto tissues thought to be derived during embryogenesis from a differentembryonal layer. For instance, endothel cells or their precursorsdetected in humans or animals that underwent marrow transplantation areat least in part derived from the marrow donor (Takahashi, Nat Med 5:434B, 1999; Lin, Clin Invest 105:71 7, 2000). Thus, visceral mesoderm andnot splanchnic mesoderm, such as MSC, derived progeny are transferredwith the infused marrow. Even more surprising are the reportsdemonstrating both in rodents and humans that hepatic epithelial cellsand biliary duct epithelial cells are derived from the donor marrow(Petersen, Science 284:1168 1170, 1999; Theise, Hepatology 31:235 40,2000; Theise, Hepatology 32:11 6, 2000). Likewise, three groups haveshown that neural stem cells can differentiate into hemopoietic cells.Finally, Clarke et al. reported that neural stem cells injected intoblastocysts can contribute to all tissues of the chimeric mouse (Clarke,Science 288:1660 3, 2000).

Transplantation of tissues and organs generated from heterologousembryonic stem cells requires either that the cells be furthergenetically modified to inhibit expression of certain cell surfacemarkers, or that the use of chemotherapeutic immune suppressors continuein order to protect against transplant rejection. Thus, althoughembryonic stem cell research provides a promising alternative solutionto the problem of a limited suppler of organs for transplantation, theproblems and risks associated with the need for immunosuppression tosustain transplantation of heterologous cells or tissue would remain. Anestimated 20 immunologically different lines of embryonic stem cellswould need to be established in order to provide immunocompatible cellsfor therapies directed to the majority, of the population (Wadman, M.,Nature (1999) 398: 551). Using cells from the developed individual,rather than an embryo, as a source of autologous or allogeneic stemcells would overcome the problem of tissue incompatibility associatedwith the use of transplanted embryonic stem cells, as well as solve theethical dilemma associated with embryonic stem cell research.

A method for differentiating mammalian stem cells according to oneembodiment can include (a) preparing a medium containing mammalian stemcells and placing the medium in a system according embodiments, (b)adding to the medium an effective amount of an agent which causesdifferentiation of the cells, producing differentiated cells, (c)contacting the cells from step (b) with an effective amount of an agentthat causes stabilization of cells produced in step (b) and (d)recovering stabilized, differentiated cells.

A method for generating differentiated cells from mammalian mesenchymalstem cells according to one embodiment can include (a) placing themesenchymal stem cells in a system according embodiments, (b) incubatingthe mesenchymal stem cells under conditions that induce the mesenchymalstem cells to differentiate and (c) recovering the differentiated cells.

A method of producing a genetically engineered cell such as stem cellsaccording to one embodiment can include (a) placing cells such as stemcells in a system according embodiments under conditions that do notcause the cells to differentiate, (b) transfecting the cells such asstem cells with a DNA construct including at least one gene of interest,(c) selecting for expression of the gene of interest in the cells suchas stem cells and (d) culturing the cells such as stem cells selected instep (c).

A method of in vivo administration of a protein or gene of interestaccording to one embodiment can include (a) placing cells such as stemcells in a system according embodiments, (b) transfecting the cells suchas stem cells with a vector including DNA or RNA that expresses aprotein or gene of interest, (c) selecting for expression of the proteinor gene of interest in the cells such as stem cells and (d) deliveringthe cells such as stem cells selected in step (c) to a mammal in needthereof.

A method of testing the ability of a candidate agent to modulate theproliferation of a lineage uncommitted cell according to one embodimentcan include (a) placing stem cells in a system according embodiments,(b) culturing the stem cells in a growth medium that maintains the stemcells as lineage uncommitted cells, (c) adding the candidate agent tothe medium and (d) determining the proliferation and lineage of thecells by mRNA expression, antigen expression or other means.

A method of preparing a stem cell matrix for use in tissue or organrepair according to one embodiment can include (a) admixing apreparation including stem cells with a physiologically acceptablematrix material to form a stem cell matrix and (b) incubating the stemcell matrix in a system according embodiments prior to use in tissue ororgan repair or treatment.

A method of tissue or organ repair or treatment, according to oneembodiment can include (a) preparing a stem cell matrix by admixing apreparation including stem cells with a physiologically acceptablematrix, (b) incubating the stem cell matrix in a system according toembodiments prior to use and (c) introducing the stem cell matrix into apatient in need thereof.

In accordance with another embodiment, mammalian stem cells can bepluripotent or multipotent stem cells or totipotent stem cells. Inaccordance with another embodiment, stem cells can be homogeneous stemcells or heterogeneous stem cells. In accordance with anotherembodiment, stem cells can be autologous or allogeneic to a recipient ora mammal.

In accordance with another embodiment, a physiologically acceptablematrix material can be selected from the group including or consistingof small intestine submucosa (SIS), crosslinked alginate, hydrocolloid,collagen, polyglycolic acid (PGA, polyglactin (PGL), fleeces, silk,keratin, dead de-epidermized skin equivalents, polyesters,polyalkylenes, polyfluoroethylenes, polyvinyl chloride (PVC),polystyrene, polysulfones, cellulose acetate, glass fibers, and inertmetal fibers.

In accordance with another embodiment, stem cells can be obtained from atissue selected from the group consisting of adult, embryonic, and fetaltissue. In accordance with another embodiment, such tissue can includebone marrow, muscle, adipose, liver, heart, lung, or nervous systemtissue.

In accordance with another embodiment, a stem cell matrix can be usedfor wound healing, surgical incision repair, tissue augmentation, organaugmentation, smooth muscle repair, non-smooth muscle repair, or bloodvessel repair or the like.

In accordance with another embodiment, stem cells are affixed to aphysiologically acceptable matrix material using a biological adhesivesuch as fibrin glue. In accordance with another embodiment, the fibringlue can be supplemented with at least one agent. In accordance withanother embodiment, a physiologically acceptable matrix material can beabsorbable or non-absorbable.

A method for producing a protein of interest according to one embodimentcan include (a) culturing a host cell in a system according to disclosedembodiments under conditions in which the protein is expressed; and (b)recovering the protein. A method for maintaining a culture of cellsaccording to one embodiment can include placing the cells in a systemaccording to disclosed embodiments under conditions in which the cellsremain substantially unchanged for an extended period of time. Inaccordance with another embodiment, an isolated culture of cells can beprepared according to disclosed embodiments of methods.

In accordance with another embodiment, immune cells (e.g., killer cells,T-cells or the like) can be used in various fluids as described hereinor used in systems 1, 1101. Further, immune cells can be used in variousfluids during preparation or differentiation of stem cells according tomethods otherwise known in the art. Such immune cells can be used toclean or decontaminate contaminated cells. Further, such cells can beused to detect otherwise undetectable contaminants such as fungi ormold. In addition, according to embodiments, the immune cells canimmunize or destroy contaminants such as mycoplasma, fungi, bacteria orthe like. Accordingly, the effectiveness of biological embodimentsdisclosed herein can be increased.

Embodiments of the system may be applied at any or all the stages ofcardiovascular disease, such as the early to late stages and spanningthrough drug treatment to tissue and cell regeneration. The early stagesare often treated medically with drugs and biomolecules that can bescreened and tested, discovered and developed using the technology. Thenext stages are often treated with drug-eluting stents where in thisexample, the ‘drug-elutant’ can be discovered, screened, and tested,discovered and developed using embodiments of the system. The latestages often require arterial bypass where embodiments of the system canbe used to produce tissue regenerated or engineered products, forexample arteries, from patient cells such as, for example, stem cells orprogenitor cells with various possible scaffolds such as a ePTFE orcollagen composites.

Embodiments of the system may also be used as a vascular trainer, torecondition a vein or artery under various dynamic conditions, withvarious growth factors or genetic or chemical treatment and otheradditives enhancing the therapeutic outcome of such a conditioningenvironment. This may also be also useful in reviving cryogenicallypreserved specimens.

The invention also provides embodiments of a system and a method bywhich appropriate mechanical environments are applied ex vivo to directthe remodeling of small, excised blood vessels to createtissue-engineered vessels characterized by increased length, internaldiameter, and wall thickness. Thus, the small excised vessels, arteries,or even veins, become tissue-engineered blood vessels for use invascular surgery. Embodiments of the invention further provide anevaluation of the performance of these tissue-engineered blood vesselsin vivo.

Embodiments of the system allow investigations of the hypothesis thatlongitudinal stress or strain induces artery elongation. In addition,while there are autologous donor arteries with proper diameter and wallthickness for vascular grafts, they often are of an insufficient lengthto meet the required need. For example, the internal thoracic artery hasexcellent long-term patency, but is of an adequate length for only asingle bypass graft. However, recognizing that if the artery could beelongated, it could be used to bypass multiple occlusions, and the useof vessels demonstrating inferior performance could be avoided,embodiments of the invention advantageously provides reliabletissue-engineered blood vessels of sufficient length to meet his need.

In addition, embodiments of the system are further used to explore themolecular regulation of mechanically induced vascular remodeling bycharacterizing the expression and regulation of key regulatory factors,for which the spatial expression and distribution of mRNA and proteinare monitored as a result of various mechanical loads.

Thus, embodiments of the invention also provide a protocol by whichlocalized intravascular and extravascular pressures are measured in realtime, and the measured pressures are compared with the calculatedpressure estimates.

The graft tissue component of the vascular graft may be derived fromessentially any biological tissue of interest provided the tissue hasthe proper geometrical dimensions and/or configurations for its intendedapplication. Typically, the graft tissue will be comprised of vasculartissue removed from a human or from an animal species, e.g., bovine,porcine, ovine, equine, canine, goat, etc., and may be removed fromvarious anatomical positions within the body. For example, the grafttissue may be derived from carotid arteries, thoracic arteries, mammaryarteries, and the like. The graft tissue must have a structure, e.g., atubular structure, which defines an interior lumen having dimensionssufficient for allowing blood to flow therethrough followingimplantation.

The primary component of the biological tissues used to fabricatebioprostheses is collagen, a generic term for a family of relatedextracellular proteins. Collagen molecules consists of three chains ofpoly (amino acids) arranged in a trihelical configuration ending innon-helical carboxyl and amino termini. These collagen moleculesassemble to form microfibrils, which in turn assemble into fibrils,resulting in collagen fibers. The amino acids which make up the collagenmolecules contain side groups, including amine, acid and hydroxylgroups, in addition to the amide bonds of the polymer backbone, all ofwhich are sites for potential chemical reaction on these molecules.

Because collagenous tissues degrade very rapidly upon implantation, itis preferable to stabilize the tissue if it is to be implanted into aliving system. The tissue can be stabilized using embodiments of thesystem of the invention in combination with any of a variety ofconventional approaches. For example, chemical stabilization by tissuecross-linking, also referred to as tissue fixation, can be achievedusing bi-functional and multi-functional molecules having reactivegroups capable of forming irreversible and stable intramolecular andintermolecular chemical bonds with the reactive amino acid side groupspresent on the collagen molecules. An additional method for thefixation/stabilization of the graft tissues involves a photooxidationprocess.

Such photooxidation may be carried out according to conventionalmethodologies. Suitable photooxidation process have been described, forexample in U.S. Pat. No. 5,854,397, the disclosure of which isincorporated herein by reference, and in Moore et al. (1994). Thephotooxidation process provides an efficient and effective method forcross-linking and stabilizing various proteinaceous materials including,but not limited to, collagen, collagen fibrils and collagen matrices.The term proteinaceous material as used herein includes both proteinssuch as collagen and protein-containing materials such as tissues. Thematerial to be cross-linked is generally provided as a vascular tissuesample. Such materials are harvested from the donor animal andimmediately immersed in cold buffered saline for storage, with frequentrinses and/or changes with fresh saline, until a fixation process isperformed.

The vascular tissue material to be photooxidized is then immersed,dispersed, or suspended (depending upon its previous processing) in anaqueous media for processing. Suitable media for immersion of thematerial (for purposes of convenience, the word “immersion” shaft beconsidered to include suspension and/or solubilization of theproteinaceous material) include aqueous and organic buffer solutionshaving a neutral to alkaline pH, preferably a pH of about 6.5 and abovebecause of the denaturation caused by add pH. Particularly preferred arebuffered aqueous solutions having a pH of from about 6.8 to about 8.6.

In a preferred photooxidation process, two media solutions are utilizedfor what is referred to herein as “preconditioning” the vascular tissuematerial before irradiation. The material is “preconditioned” in thesense that tissue soaked in the first media solution and irradiated inthe second are apparently better cross-linked, e.g., they show improvedmechanical properties and decreased susceptibility to proteolyticdegradation. The efficacy of this preconditioning is affected by theosmolality of the first media solution, it being preferred thatsolutions of high osmolality be used as the first media solution.Particularly preferred are sodium potassium, or organic buffer solutionssuch as sodium, chloride, sodium phosphate, potassium chloride,potassium phosphate, and Good's buffers having a pH of from about 6.8 toabout 8.6, the osmolality of which have been increased by addition of asolute such as 4M sucrose or other soluble, high molecular weightcarbohydrate to between about 393 mosm and about 800 mosm.

The solute added to increase the osmolality of the first media may havean adverse effect on the degree of cross-linking of the product whenpresent during irradiation. Consequently, after soaking in the firstmedia, the tissue is preferably removed therefrom and immersed in asecond media for irradiation. The second media is preferably an aqueousbuffered solution having a pH of from about 6.8 to about 8.6 in whichthe photo-catalyst is dissolved. Preferred second media are sodium andpotassium phosphate buffers having a pH of from about 7.4 to about 8.0and an osmolality of from about 150 to about 400 mosm.

The tissue may be advantageously immersed sequentially in the firstmedia and then in the catalyst-incorporated second media prior tophotooxidation for a total period of time sufficient to allow tissue,dye, and medium to reach equilibrium. When the ratio of theconcentration of the medium to that of the material to be cross-linkedis in the range of from about 10:1 to 30:1, equilibrium can generally bereadily achieved. The ratio of the concentrations is generally notcritical, and may be adjusted up or down as desired. Once an equilibriumis reached, the sample is photooxidized in the catalyst-incorporatedmedium. The time required to reach equilibrium varies depending uponsuch factors as, for instance, the temperature of the media solutions,the osmolality of the first media, and the thickness of the tissue orother sample of proteinaceous material. A period of time as short as afew minutes or as long as several days may be sufficient, but it hasbeen found that periods of from minutes to hours duration is generallysufficient to allow sufficient time for most collagenous materials andmedia to equilibrate.

The catalysts for use in the photofixation process includephotooxidative catalysts (photo-catalysts) that when activated willcause transfer of electrons or hydrogen atoms and thereby oxidize asubstrate in the presence of oxygen. Although varied results arepossible depending upon the particular catalyst utilized, appropriatecatalysts include, but are not limited to, those listed in Oster, etal., J. Am. Chem. Soc. 81: 5095, 5096 (1959). Particularly preferredcatalysts include methylene blue, methylene green, rose bengal,riboflavin, proflavin, fluorescein, eosin, and pyridoxal-5-phosphate.

The concentration of catalyst in the media will vary based on severalprocess parameters, but should be sufficient to insure adequatepenetration into the material to be cross-linked and to catalyze thephotooxidation of the protein. A typical catalyst concentration rangesfrom about 0.0001%-0.25% (wt/vol); the preferred concentration rangesfrom about 0.001 to about 0.01%.

To achieve maximum cross-linking and stabilization of the vasculartissue, the following steps may be taken: (1) the photooxidativecatalyst should be completely solubilized in the reaction medium priorto use to ensure that the desired dye concentration is achieved; (2) theconcentration of the catalyst in the tissue or suspension should be inequilibrium with that in the surrounding medium; and (3) the catalystsolution should be filtered to remove any sizable particulate matter,including chemical particulates, therefrom.

Because the photofixation process involves primarily an oxidativereaction, to assure completion of the reaction, an adequate supply ofoxygen must be provided during photooxidation. While an oxygenconcentration of about 20% by volume (referring to the concentration ofoxygen in the atmosphere over the media) is preferred to assuresufficient dissolved oxygen in the media to prevent oxygen content frombecoming rate limiting, all concentrations >0% can also be used.Depending upon the temperature at which the material is held duringexposure to light, the oxygen requirement can be met, for instance, byagitating the solution or otherwise mixing the solution, suspension, orsample during the reaction process. Oxygen concentration in theatmosphere over the media during irradiation is preferably maintained inthe range of from about 5% to about 40%. Such concentrations (againdepending upon temperature) can also be achieved, for instance, bybubbling air into the media during irradiation of the tissue or, ifconcentrations higher than about 20% are desired, by bubbling oxygenmixtures or air having an increased oxygen content into the media.

As with other catalytic or kinetic-type reactions, the temperature atwhich the reaction is run directly affects the reaction rate and theoxygen available in the media. Tests conducted with various mediaranging in pH from about 6.8 up to about 7.4 indicate that as thetemperature of the media increases from about 4 C to about 50 C., oxygenconcentration drops in roughly linear fashion from about 11-12 ppm toabout 5 ppm. The dye-catalyzed photooxidation process is exothermic, andit is, therefore, preferred that a relatively constant temperature bemaintained during irradiation of the proteinaceous material to preventdenaturation of the proteinaceous material and the driving of the oxygenout of the media by the increase in temperature. Usually, arecirculating bath is sufficient to maintain and control the temperaturewithin the jacketed reaction vessel or chamber but placement of thereaction chamber within a controlled environment such as a refrigeratoror freezer will work as well. As disclosed herein, photooxidationconducted at temperatures ranging from about −2 C to 40 C. has beenshown to be effective; the preferred temperatures being about 0 C toabout 25 C. To prevent or alleviate denaturation of the proteincomprising the vascular tissue, temperatures below the denaturationtemperature of that protein are preferred. Likewise, temperatures abovethe freezing point of the reaction medium are also preferred.

The process is conducted at temperatures low enough to avoid heatdenaturation and pH high enough to avoid acid denaturation of thecollagen or other proteinaceous material during cross-linking. Likewise,temperature is held at a level sufficient to maintain the oxygenconcentration in the media in which the proteinaceous material isimmersed during irradiation.

Once the tissue is prepared, it is photo-irradiated, preferably in acontrolled system wherein temperature, distance to light source,irradiation energy and wavelength, oxygen concentration and period ofirradiation can be monitored and/or maintained. The tissue isphoto-irradiated under conditions sufficient to cause cross-linking.Photooxidation is generally achieved using incandescent, white light orfluorescent light, i.e., visible light, or that portion of light in thevisible range that is absorbed by the catalyst.

The intensity of the light employed, and the length of time required tocross-link a given proteinaceous material will vary depending uponseveral factors. These include: (1) the type and amount of proteinaceousmaterial; (2) the thickness of the tissue sample; (3) the distancebetween the proteinaceous material and the irradiation source; (4) thecatalyst employed; (5) the concentration of catalyst; and (6) the typeand intensity of the light source. For instance, exposure time may varyfrom as little as a few seconds up to as much as about 160 hours. Withregard to the intensity of the light, one or more lights may be used ofintensity preferably ranging up to about 150 watts, preferably held at adistance from about 2.5 cm to 12 cm from the sample surface. Greaterexposure time is required when fluorescent or lower power lights areutilized. These ranges are quite variable; however, they may be easilydetermined for a given material without resort to undue experimentation.

Evidence of the cross-linking of the vascular tissue by photooxidationmay be provided by several approaches. For instance, polyacrylamide gelelectrophoresis of the irradiated material in sodium dodecylsulfate (forexample, 0.1%) evidences such cross-linking by a significant decrease inthe amount of lower molecular weight material with the simultaneousappearance of high molecular weight material.

Further evidence of cross-linking may be provided by known solubilityand digestibility tests. For instance, cross-linked collagen isgenerally insoluble such that solubility tests provide direct evidenceof the degree of cross-linking. The digestibility tests involveincubation of the proteinaceous product with a proteolytic enzyme suchas papain, trypsin, pepsin, or bacterial collagenase, and the subsequenttesting of the media in which the product and enzyme are incubated forsoluble degradation products of the cross-linked product. The test isgenerally accomplished by pelletizing the undigested, cross-linkedtissue by centrifugation and testing the resulting supernatant fordegradation products.

Following photo-irradiation, the cross-linked product may beadvantageously subjected to additional treatments for the removal of thecatalyst and other chemicals or impurities found therein before beingused as a vascular graft. Multiple rinses in a fresh buffer solution,for example, may be used, followed by at least partial removal of waterby treatment with, for instance, ethanol. The number of rinses and thevolume of rinse solution required depend upon the mass of the tissue andthe catalyst concentration utilized.

In addition to the use of photooxidation processes for the fixation ofthe graft tissue, numerous other fixation methods have been describedand are readily available in the art and may be used in conjunction withembodiments of the invention. For example, glutaraldehyde, and otherrelated aldehydes, have seen widespread use in preparing cross-linkedbiological tissues. Methods for glutaraldehyde fixation of biologicaltissues have been extensively described and are well known in the art.In general, a tissue sample to be cross-linked is simply contacted witha glutaraldehyde solution for a duration effective to cause the desireddegree of cross-linking within the biological tissue being treated.

Many variations and conditions have been applied to optimizeglutaraldehyde fixation procedures. For example, lower concentrationshave been found to be better in bulks tissue cross-linking compared tohigher concentrations. It has been proposed that higher concentrationsof glutaraldehyde may promote rapid surface cross-linking of the tissue,generating a barrier that impedes or prevents the further diffusion ofglutaraldehyde into the tissue bulk. For most bioprosthesisapplications, the tissue is treated with a relatively low concentrationglutaraldehyde solution, e.g., typically between 0.1%-5%, for 24 hoursor more to ensure optimum fixation. Of course, various othercombinations of glutaraldehyde concentrations and treatment times willalso be suitable depending on the objectives for a given application.

In addition to bifunctional aldehydes, many other chemical fixationprocedures have been described (for review, see Khor, Biomaterials 18:95-105, 1997). For example, some such methods have employed polyethers,polyepoxy compounds, diisocyanates, azides, etc. These and otherapproaches available to the skilled individual in the art for treatingbiological tissues wills be suitable for cross-linking vascular grafttissue in embodiments of systems according to his invention.

The hemodynamic forces recreated within and by embodiments of the systemmay also be used to improve organ transplant procedures and make theseprocedures more successful by providing an appropriate environments(e.g., hemodynamic) for an organ prior to transplant, both during thetransport period and while awaiting actual transplant. Moreparticularly, providing a simulated pulsatile or hemodynamicenvironment, which may represent in vivo conditions of the particularorgan, to the organ during these periods protects the integrity of theorgan by maintaining its proper functionality after it has been removedso as to provide the best possible transition and adaptation in a newhost. Also, embodiments of the system may be used to re-vive an organthat was cryopreserved or treated with a type of preservation treatmentas well.

The invention also provides an embodiment of a system for applyingcontrolled shear flow stress to mammalian cell cultures used forartificial cartilage production.

Applying shear flow stress to a three-dimensional or monolayerchondrocyte culture advantageously increases the ratio of type II totype I collagen produced by the chondrocytes. The shear flow stress alsoadvantageously enhances maintenance of the chondrocyte phenotype. Thus,application of shear flow stress according to embodiments of thisinvention improves the functional outcome of a three-dimensional ormonolayer chondrocyte culture and increases the useful lifetime of themonolayer culture.

Applying shear flow stress to stem cells induces or promotesdifferentiation of the stem cells into chondrocytes. Inducing orpromoting stem cells to differentiate into chondrocytes is accomplishedby substituting stem cells for chondrocytes in the shear flow methoddescribed herein with regard to chondrocytes. The chondrocytes arisingfrom the stem cell differentiation process are maintained in theculture, under shear flow stress, for a sufficient time to allowproduction of artificial cartilage.

Shear flow stress also can be used according to embodiments of thisinvention to induce transdifferentiation of differentiated cells intochondrocytes. Transdifferentiation is accomplished by substituting,differentiated cells other than chondrocytes, e.g., myoblasts orfibroblasts, in the shear flow method described herein with regard tochondrocytes. In response to the shear flow stress, the differentiatedcells transdifferentiate into chondrocytes. The chondrocytes arisingfrom the transdifferentiation process are maintained in the culture,under shear flow stress, for a sufficient time to allow production ofartificial cartilage.

Artificial cartilage produced according to any embodiment of thisinvention can be used for surgical transplantation, according toestablished medical procedures, to replace damaged or missing cartilage.Typically, artificial cartilage is employed in the repair of humanjoints, e.g., knees and elbows.

Preferably, the cultured chondrocytes are anchored, i.e., attached, to asubstrate, whether grown as a monolayer or grown in a 3-dimensionalculture. A monolayer-supporting surface, or a 3-dimensional scaffold, ina bioreactor is inoculated with chondrocytes, stem cells, ordifferentiated cells suitable for transdifferentiation. Artificialcartilage can be produced by growing chondrocytes in a conventionalmammalian tissue culture medium, e.g., RPMI 1640, Fisher's, Iscove's orMcCoy's. Such media are well known in the art, and are commerciallyavailable. Typically, the cells are cultured at 37 C in air supplementedwith 5% CO2. Under these conditions, a chondrocyte monolayer or a threedimensional cartilage matrix is produced in approximately 7 to 56 days,depending on the cell type used for inoculation and the cultureconditions.

Isolated chondrocytes can be used to inoculate the surface of a supportor a 3-dimensional matrix. Alternately, stem cells, or cells suitablefor transdifferentiation can be used for inoculation.

Cells used for inoculation of cultures used in the invention can beisolated by any suitable method. Various starting materials and methodsfor chondrocyte isolation are known. See generally, Freshney, Culture ofAnimal Cells. A Manual of Basic Techniques, 2d ed., A. R. Liss Inc., NewYork, pp 137-168 (1987). Examples of starting materials for chondrocyteisolation include mammalian knee joints or rib cages.

If the starting material is a tissue in which chondrocytes areessentially the only cell type present, e.g., articular cartilage, thecells can be obtained directly by conventional collagenase digestion andtissue culture methods. Alternatively, the cells can be isolated fromother cell types present in the starting material. One known method forchondrocyte isolation includes differential adhesion to plastic tissueculture vessels. In a second method, antibodies that bind to chondrocytecell surface markers can be coated on tissue culture plates and thenused to selectively bind chondrocytes from a heterogeneous cellpopulation. In a third method, fluorescence activated cell sorting(FACS) using chondrocyte-specific antibodies is used to isolatechondrocytes. In a fourth method, chondrocytes are isolated on the basisof their buoyant density, by centrifugation through a density gradientsuch as Ficoll.

Examples of tissues from which stem cells for differentiation, ordifferentiated cells suitable for transdifferentiation, can be isolatedinclude placenta, umbilical cord, bone marrow, skin, muscle, periosteum,or perichondrium. Cells can be isolated from these tissues by explantculture and/or enzymatic digestion of surrounding matrix usingconventional methods.

When the artificial cartilage construct has grown to the desired sizeand composition, a cryopreservative fluid can be introduced intoembodiments of the system. The cryopreservative fluid freezes theartificial cartilage construct for future use. Cryopreservation methodsand materials for mammalian tissue culture material are known to thoseof ordinary skill in the art.

Methods and materials for 3-dimensional cultures of mammalian cells areknown in the art. See, e.g., U.S. Pat. No. 5,266,480. Typically, ascaffold is used in a bioreactor growth chamber to support a3-dimensional culture. The scaffold can be made of any porous, tissueculture-compatible material into which cultured mammalian cells canenter and attach or anchor. Such materials include nylon (polyamides),dacron (polyesters), polystyrene, polypropylene, polyacrylates,polyvinyl chloride, polytetrafluoroethylene (teflon), nitrocellulose,and cotton. Preferably, the scaffold is a bioabsorbable or biodegradablematerial such as polyglycolic acid, catgut suture material, or gelatin.In general, the shape of the scaffold is not critical.

Optionally, prior to inoculating chondrocytes into the scaffold, stromalcells are inoculated into the scaffold and allowed to form a stromalmatrix. The chondrocytes are then inoculated into the stromal matrix.The stromal cells can include fibroblasts. The stromal cells can alsoinclude other cell types.

A 3-dimensional culture can be used in a system of the invention andshear flow stress applied to the chondrocytes by the movement of theliquid culture medium pumped through the growth chamber, which containsthe 3-dimensional culture. Preferably, in such embodiments, the scaffoldand attached cells are static.

Embodiments of the system for simulating hemodynamic forces as embodiedand broadly described herein is capable of generating the complete rangeof hemodynamic force patterns in the interest and advancement ofcardiovascular research, and will make new avenues of research anddevelopment available which were never before possible, at any cost.Embodiments of systems and methods will greatly advance ourunderstanding of cardiovascular function and disease, and allowpharmacologic and genetic strategies to be tested at much lower coststhan conventional methods of experimentation. Ultimately, patients willbenefit the most, since embodiments of the invention will advance newconcepts in cardiovascular disease progression, development, andtreatment. Healthy patients can function as productive members ofsociety, improve their quality of life, and reduce the cost of medicaltreatment.

Hemodynamic conditions are one class of dynamic conditions (FIG. 18) andaffect cardiovascular physiology and pathology. Pulsatile flow (Q),pressure (P), and diameter (D) waveforms exert wall shear stress (WSS),normal stress, and circumferential strain (CS) (types of dynamicconditions as shown in FIG. 17) on blood vessels. In vitro studies todate have focused on either WSS or CS but not their interaction. Studiescaused at using embodiments of systems 1 and 1101 have demonstrated thatconcomitant WSS and CS affect endothelial cell (EC) biochemical responsemodulated by the temporal phase angle between WSS and CS (stress phaseangle, SPA) (one type of dynamic condition as shown in FIG. 17). Systems1, 1101 have shown that large negative SPA occurs in regions of thecirculation where atherosclerosis and intimal hyperplasia are prevalent,and that nitric oxide (NO) biochemical secretion was significantlydecreased in response to a large negative SPA of −180 deg with respectto an SPA of 0° in bovine aortic endothelial cells (BAEC) at 5 hr.Systems 1, 1101 use the discrete hemodynamic conditions ofpro-atherogenic (SPA=−180 deg) and normopathic (SPA=0 deg) states asinput information to study the physiologic SPA used to produce thecorresponding hemodynamic conditions at the tubular structures.Accordingly, systems 1, 1101 demonstrate that one type of dynamiccondition (SPA) plays an important role in hemodynamics with respect tovascular remodeling, homeostasis, and pathogenesis, and that a largenegative SPA is pro-atherogenic.

Endothelial cells (EC) lining all blood vessel walls serve as sensorsand transducers of two types of dynamic conditions, namely, wall shearstress (WSS) and circumferential strain (CS), in the class ofhemodynamic conditions. WSS and CS (also referred to as stretch)independently influence EC morphology and biochemistry. See, forexample, Davies, P. F. et al., 2001, “Hemodynamics and the Focal Originof Atherosclerosis: A Spatial Approach to Endothelial Structure, GeneExpression, and Function,” Ann. N.Y. Acad. Sci., 947, pp. 7-16; 947, pp.16-17; Kito, H. et al., 1998, “Cyclooxygenase Expression in BovineAortic Endothelial Cells Exposed to Cyclic Strain,” Endothelium, 6(2),pp. 107-112 and Frangos, S. G. et al., 2001, “The Integrin-MediatedCyclic Strain-Induced Signaling Pathway in Vascular Endothelial Cells,”Endothelium, 8(1), pp. 1-10, the contents of which are incorporatedherein by reference.

WSS and CS also independently influence EC monolayer permeability tomacromolecules and water. See, for example, Sill, H. W. et al., 1995,“Shear Stress Increases Hydraulic Conductivity of Cultured EndothelialMonolayers,” Am. J. Physiol., 268 (2 Pt 2), pp. H535-H1543 and Lever, M.J., Tarbell, J. M., and Caro, C. G., 1992, “The Effect of Juminal Flowin Rabbit Carotid Artery on Transmural Fluid Transport,” Exp. Physiol.,77(4), pp. 553-563, the contents of which are incorporated herein byreference. WSS is an important fluid mechanical mediator ofatherosclerosis and together with CS is important in vascular regulationand remodeling. See, for example, Gimbrone, Jr., M. A., 1999, “VascularEndothelium, Hemodynamic Forces, and Atherogenesis,” Am. J. Pathol.,155(1), pp. 1-5, the contents of which are incorporated herein byreference.

Changes in flow rate are sensed by the endothelium through the WSS byreleasing vasoactive agents that modulate smooth muscle contraction ordilation as discussed for example in Furchgott, R. F., and Zawadzki, J.V., 1980, “The Obligatory Role of Endothelial Cells in the Relaxation ofArterial Smooth Muscle by Acetylcholine,” Nature (London), 288(5789),pp. 373-376; Kohler, T. R., and Jawien, A., 1992, “Flow AffectsDevelopment of Intimal Hyperplasia After Arterial Injury in Rats,”Arterioscler. Thromb., 12(8), pp. 963-971 and Cooke, J. P. et al., 1990,“Flow Stimulates Endothelial Cells to Release a Nitrovasodilator That isPotentiated by Reduced Thiol,” Am. J. Physiol., 259(3 Pt 2), pp.H804-H812, the contents of which are incorporated herein by reference.Different mechanical environments give rise to different endothelialcell phenotypes throughout the circulation. See, for example, Chappell,D. C. et al., 1998, “Oscillatory Shear Stress Stimulates AdhesionMolecule Expression in Cultured Human Endothelium,” Circ. Res., 82, pp.532-539 and Nerem, R. M. et al., 1998, “The Study of the Influence ofFlow on Vascular Endothelial Cell Biology,” Am. J. Med. Sci., 316, pp.169-175, the contents of which are incorporated herein by reference.

Vascular smooth muscle cells also experience hemodynamic forces thathave been implicated in their proliferation and migration as observed inatherosclerosis. See, for example, Kohler, T. R., and Jawien, A., 1992,“Flow Affects Development of Intimal Hyperplasia After Arterial Injuryin Rats,” Arterioscler. Thromb., 12(8), pp. 963-971 and Kohler, T. R.,and Jawien, A., 1992, “Flow Affects Development of Intimal HyperplasiaAfter Arterial Injury in Rats,” Arterioscler Thromb., 12(8), pp.963-971, the contents of which are incorporated herein by reference.Most studies that examined simultaneous WSS and CS have not controlledor had limited control of the temporal phase angle between WSS and CS(stress phase angle, SPA). See, for example, Zhao, S. et al., 1995,“Synergistic Effects of Fluid Shear Stress and Cyclic CircumferentialStretch on Vascular Endothelial Cell Morphology and Cytoskeleton,”Arterioscler., Thromb., Vasc. Biol., 15(10), pp. 1781-1786; Benbrahim,A. et al., 1994, “A Compliant Tubular Device to Study the Influences ofWall Strain and Fluid Shear Stress on Cells of the Vascular Wall,” J.Vasc. Surg., 20(2), pp. 184-194; Ziegler, T. et al., 1998, “Influence ofOscillatory and Unidirectional Flow Environments on the Expression ofEndothelin and Nitric Oxide Synthase in Cultured Endothelial Cells,”Arterioscler., Thromb., Vasc. Biol., 18(5), pp. 686-692 and Qiu, Y., andTarbell, J. M., 2000, “Interaction Between Wall Shear Stress andCircumferential Strain Affects Endothelial Cell Biochemical Production,”J. Vasc. Res., 37(3), pp. 147-157, the contents of which areincorporated herein by reference.

Nitric oxide (NO) is one of the smallest biomolecules produced inmammalian cells and plays a major role in vascular homeostasis, asdiscussed, for example, in Ignarro, L. J., 1990, “Nitric Oxide. A NovelSignal Transduction Mechanism for Transcellular Communication,”Hypertension, 16(5), pp. 477-483, the contents of which are incorporatedherein by reference. The content longitudinal and/or radialvelocity/flow concentration of NO are types of dynamic conditions FIG.17). The small size of NO permits unhindered movement to neighboringcells, however, the short half-life (<5 seconds) limits its range. Redblood cells can aid in the transport of NO through binding withhemoglobin to form nitrosyl-heme adducts that are more stable than freeNO. NO production occurs trough a redox reaction involving threecosubstrates, five cofactors, and nitric oxide synthase (NOS) that leadsto the conversion of L-arginine to L-citruline and release of NO. See,for example, Nathan, C., and Xie, Q. W., 1994, “Nitric Oxide Synthases:Roles, Tolls, and Controls,” Cell, 78(6), pp. 915-918 and Nathan, C.,and Xie, Q. W., 1994, “Regulation of Biosynthesis of Nitric Oxide,” J.Biol. Chem., 269(19), pp. 13725-13728, the contents of which areincorporated herein by reference.

Three iso-forms of NOS exist: nNOS—predominant in neuronal cells;iNOS—constitutive expression and mainly in found in macrophages; andeNOS—located in endothelial cells and the only isoform to form amembrane-bound linkage in the signal-transducing domains of theplasmalemma, the caveolae. See, for example, Bevan, J. A., and Siegel,G., 1991, “Blood Vessel Wall Matrix Flow Sensor: Evidence andSpeculation,” Blood Vessels, 28(6), pp. 557-556; Bevan, J. A., andLaher, I., 1991, “Pressure and Flow-Dependent Vascular Tone,” FASEB J.,5(9), pp. 2267-2273; Bevan, J. A., 1991, “Pressure and Flow: Are Thesethe True Vascular Neuroeffectors?” Blood Vessels, 28(1-3), pp. 164-172;Davies, P. F., 1995, “Flow-Mediated Endothelial Mechanotransduction,”Physiol. Rev., 75(3), pp. 519-560; Gimbrone, Jr., M. A. et al., 2000,“Endothelial Dysfunction, Hemodynamic Forces, and Atherogenesis,” Ann.N.Y. Acad. Sci., 902, pp. 230-239; 902, pp. 239-240 and Cahill, P. A. etal., 1996, “Increased Endothelial Nitric Oxide Synthase Activity in theHyperemic Vessels of Portal Hypertensive Rats,” J. Hepatol, 25(3), pp370-378, the contents of which are incorporated herein by reference. WSSincreases NO secretion. See for example, Cahill, P. A. et al., 1996,“Increased Endothelial Nitric Oxide Synthase Activity in the HyperemicVessels of Portal Hypertensive Rats,” J. Hepatol, 25(3), pp. 370-378,the contents of which are incorporated herein by reference. CS alone andCS combined with WSS also augment NO release. See, for example, Awolesi,M. A., Sessa, W. C., and Sumpio, B. E., 1995, “Cyclic StrainUp-regulates Nitric Oxide Synthase in Cultured Bovine Aortic EndothelialCells,” J. Clin. Invest., 96(3), pp. 1449-1454, the contents of whichare incorporated herein by reference.

Pulsatile blood flow in the arterial circulation produces oscillatorywall shear stress with mean values from 5 to 40 dyne/cm². See, forexample, Lipowsky, H. H., 1995, “Shear Stress in the Circulation,” inFlow-dependent Regulation of Vascular Function, edited by J. A. Bevan etal., the contents of which are incorporated herein by reference.Pulsatile blood pressure causes large arteries to expand predominantlyin the circumferential direction, whereas longitudinal expansion isconstrained by blood vessel branching and tethering. See, for example,Dobrin, P. B., 1978, “Mechanical Properties of Arteries,” Physiol. Rev.,58, pp. 397-460, the contents of which are incorporated herein byreference. As the vessel expands, a uniform circumferential strain isproduced. For this reason, a three-dimensional geometry tube or tubularstructure, instead of a two-dimensional flat membrane, is used insystems 1, 1101, which produces heterogeneous strain fields. See, forexample, Brown, T. D., 2000, “Techniques for Mechanical Stimulation ofCells in Vitro: A Review,” J. Biomech., 33, pp. 3-14, the contents ofwhich are incorporated herein by reference. In one embodiment of theinvention, systems 1, 101, 1101 produce a maximum cyclic strain ordiameter variation, CS=(D_(max)−D_(min))/D_(mean), driven by pulsingtransmural pressure in large arteries such as the thoracic aorta,carotid artery, femoral artery, and pulmonary artery ranges from 2% to18% over the pressure pulse. The venous systemic circulation has almostno diameter variation due to the low pressure pulse. Atherosclerosisoccurs in the large arteries where CS is significant. Accordingly in oneembodiment of the invention, systems 1, 1101 produces both hemodynamicconditions CS and WSS.

Blood vessel endothelial cells in vivo are subjected to simultaneouspulsatile CS and WSS acting approximately in perpendicular directions.The temporal phase angle between pressure and flow (e.g., impedancephase angle, IPA also a type of dynamic condition as per FIG. 17)generated by global wave reflection in the circulation, as well as theinertial effects of blood flow, cause temporal phase shifts to occurbetween CS and WSS. The temporal phase angle between CS and WSS (SPA) invivo generates complex, time-varying mechanical force patterns on the ECmonolayer, as shown in FIGS. 10A-10H, 25A-25C and 30-34.

Physiologic factors contribute to variations in SPA throughout thecirculation. SPA can be described as the phase angle between diameter(D) and WSS (τ), denoted as φ(D−τ), that shows CS is generallysynchronous with vessel diameter D) variation. The SPA can be decomposedinto two parts

φ(D−τ)=φ(D−Q)−φ(τ−Q)≅φ(P−Q)−φ(τ−Q)

where φ(D−Q) is approximately equal to the IPA, φ(P−Q) since diameter(D) and pressure (P) are nearly in phase for an elastic vessel ortubular structure, and φ(τ−Q) is the phase angle between the WSS andflow rate. φ(P−Q) is determined from distal resistance, compliance, andwave reflections. φ(P−Q) of the first harmonic of a physiologic waveformapproaches −45 deg (P lags Q by 45 deg) in the aorta and large arteriesthat feed high impedance flow circuits (except for coronary arteries dueto their unique flow circuit), approaches 0 deg in small arteries due toreduced distal compliance, and also approaches 0 deg in veins that feedlow impedance flow circuits. See, for example, Nichols, W. W., andO'Rourke, M. F., 1998, McDonald's Blood Flow in Arteries Theoretical,Experimental, and Clinical Principles, Arnold and Oxford UniversityPress, New York, the contents of which are incorporated herein byreference.

φ(τ−Q), the shear-flow phase angle, in straight vessels is determined bythe relative importance of unsteady inertia and viscous forces anddepends strongly on the unsteadiness parameter [α≡α√{square root over((w/ν))}; α=vessel radius, w=fundamental frequency of the heart beat,and v=kinematic viscosity of blood. For large straight arteries andveins with high α,φ(τ−Q) approaches +45 deg and for small, straightarteries and veins with low α,φ(τ−Q) approaches 0 deg, which systems 1,1101 can produce in specimen 12 or tubular structure 1112. See, forexample, Womersley, J. R., 1955, “Method for Calculation of Velocity)Rate of Flow and Viscous Drag in Arteries When the Pressure Gradlent isKnown,” J. Physiol. (London), 127, pp. 553-563, the contents of whichare incorporated herein by reference.

Based on the above discussion, the following SPA approximations instraight vessels can be summarized and produced by systems 1, 1101:

Large artery (straight): φ(D−τ)=−45 deg−45 deg=−90 deg

Large vein (straight): φ(D−τ)=0 deg−45 deg=−45 deg

Small artery (straight): φ(D−τ)=0 deg−0 deg=0 deg

Small vein (straight): φ(D−τ)=0 deg−0 deg=0 deg

The shear-flow phase angle is strongly dependent on local vesselgeometric factors that can induce spatial skewing of velocity profilesand flow separation. This can lead to local spatial distribution of SPA(a type of dynamic condition as shown in FIG. 17) in certain vesselssuch as those associated with intimal hyperplasia and atherosclerosis.Several high-risk arterial geometries include the aortic abdominalbifurcation (see, for example, Lee, C. S., and Tarbell, J. M., 1997,“Wall Shear Rate Distribution in an Abdominal Aortic Bifurcation Model:Effects of Vessel Compliance and Phase Angle Between Pressure and FlowWaveforms,” J. Biomech. Eng., 119(3), pp. 333-342, the contents of whichare incorporated herein by reference) curved coronary artery (see, forexample, Qiu, Y., and Tarbell, J. M., 2000, “Numerical Simulation ofPulsatile Flow in a Compliant Curved Tube Model of a Coronary Artery,”J. Biomech. Eng., 122(1), pp. 77-85, the contents of which areincorporated herein by reference), and end-to-end undersized graftanastomosis, all of which can be produced at a specimen 12 in systems 1,101, 1101. For example, in the aortic abdominal bifurcation, the SPAdrops along the outer wall, especially near the disease-prone regionopposite the flow divider to −80 deg (e.g., normal) and −100 deg (e.g.,hypertensive case). This region of complex hemodynamic conditions in theaortic abdominal bifurcation is also characterized by low shear stressas opposed to the high shear region of flow divider. The inner wall(flow divider) has a higher SPA (e.g., −20 deg normal and −55 deghypertensive) and higher shear stress. Systems 1, 1101 can produce allof these hemodynamic conditions at specimen 12 or tubular structure1112. Not only is the SPA large and negative in the region ofatherosclerotic plaque development, but also hypertension will furtherdecrease the SPA, resulting in a more atherogenic condition. Suchpathology (e.g., dynamic conditions as shown in FIG. 17) can bereproduced by systems 1, 1101.

A curved coronary artery experiences complex hemodynamics primarilycaused by the unique coronary flow circuit that allows for the mostextreme SPA in the cardiovascular circulation. The entire coronaryartery experiences a large negative SPA (e.g., SPA<−180 deg: −250 deg onthe inner wall, −220 deg on the outer wall) which can be produced atspecimen 12 or tubular structure 1121 by systems 1, 1101. Coronaryarteries are the most disease-prone arteries in the cardiovascularcirculation. In all instances, the SPA is more negative in low shearpathologic regions than in high shear healthy regions.

Thus, regions of the circulation prone to pathologic development such asatherosclerosis and intimal hyperplasia are characterized by largenegative SPA values relative to regions typically without pathologicdevelopment (e.g., veins, small arteries, high shear regions in largearteries). Accordingly, pathologic development or conditions can bemodeled using systems 1, 1101. Endothelial biomolecule production isaffected by a negative SPA (−100 deg). See, for example, Qiu, Y., andTarbell, J. M., 2000, “Interaction Between Wall Shear Stress andCircumferential Strain Affects Endothelial Cell Biochemical Production,”J. Vasc. Res., 37(3), pp. 147-157, the contents of which areincorporated herein by reference.

Detection of fluid molecules (e.g., endothelial cell NO production) candemonstrate affects of dynamic conditions of FIG. 17 (e.g., highlynegative SPA) for a class of dynamic conditions, as shown in FIG. 18(e.g., hemodynamic conditions) on EC and the cardiovascular system(e.g., coronary arteries).

The present example (e.g., FIG. 46) is provided to demonstrate thecapability and utility of the embodiments of the invention forreproducing in vivo mammalian hemodynamic conditions in vitro. Inparticular aspects, the present example will also demonstrate theexemplary utility of the equipment for obtaining a in vitro and in vivoinformation relating to classes and types of dynamic conditions (e.g.,FIGS. 17A, 17B and 18). Types of dynamic conditions g(t) that weremeasured in the present study include changes in production of NO fromECs exposed to pathologic (e.g., BAECs −180 SPA, FIG. 10B) hemodynamicconditions versus production of NO from ECs exposed to normal (e.g.,BAES, 0 deg SPA, FIG. 10A). Additional types of dynamic conditions whichsystems 1, 1001 measured and/or controlled include changes in specimens12 or tubular structure 1112 hemodynamic conditions monitored for Q(t),D(t), P(t), pH, temperature viability, (directly) and NO, WSS, CS, SPA(indirectly).

The cell culture consisted of primary bovine aortic endothelial cells(BAECs) obtained from fresh aortas. Briefly, fresh bovine aortas wereobtained and rinsed with cold HBSS and 1% penicillin-streptomycin. Theaorta was cut longitudinally along the intercostal arteries and formedinto a trough. Ten ml of collagenase (e.g., Blendzyme from RocheDiagnostics Corp.) was placed in the trough for 40 min, removed, andcentrifuged (e.g., repeated five times). The cell population purity was97%-99% as determined via labeled Dil-acetylated LDL, a common markerfor endothelial cells, and flow cytometry.

The BAECs were grown with 10% FBS (e.g., F-2442 from Sigma ChemicalCo.), MEM w/phenol red (e.g., M-0769 from Sigma Chemical Co.), 14%bovine serum albumin (e.g., BSA 30%, A-7284 from Sigma Chemical Co.), 1%penicillin-streptomycin (e.g., 50 U/mL and 50 μg/mL, P0906 from SigmaChemical Co.), and L-glutamine (2 mM) until passage (4-8) (populationdoubling 15-18). Experimental medium was phenol red free MEM+9.5%dextran (e.g., ˜148 kD, D4876 from Sigma Chemical Co.) to increaseviscosity (6.38 cP) to achieve desired shear stress. BAECs were platedon fibronectin (e.g., bovine plasma F-1141 from Sigma Chemical Co., 30μg/ml in MEM) treated silicone tubes (e.g., pretreated with 70% sulfuricacid for 10 min). The plating density was 4-6×10⁴ cells/m. The culturedtubes were grown in the incubator for 3-4 days until confluence prior toexperiment. The tube surface area was 38 cm². Each experiment consistedof pulsatile conditions (SPA=0 deg or −180 deg) with companion controls;steady shear stress (SS), static control (SC), and pressurized control(PC). The conditions were 10±10 dynes/cm², 70±20 mmHg, and 4±4% diametervariation at 1 Hz and 37° C. Direct microscope visualization verifiedcell attachment before and after experiments.

Nitric oxide (NO) measurement was performed via a fluorometric method.Indirect determination was performed via examination of NO breakdownproducts NO₃ ⁻ and NO₂ ⁻. The fluorometric quantification is based onthe reaction of nitrite with 2,3-diaminonapthalene (DAN) that producesthe fluorescent compound 1-(H)-napthotriazole and can detectconcentrations as low as 10 nM. See, for example, Stamler, J. S., 1995,“S-Nitrosothiols and the Bloregulatory Actions of Nitrogen OxidesThrough Reactions With Thiol Groups,” Curr. Top. Microbiol. Immunol.,196, pp. 19-36, the contents of which are incorporated herein byreference. Next, 10 μl of DAN solution (0.05 mg/ml in 0.62 M HCl) wasadded to each well and refrigerated at 4° C. for 10 min and the reactionwas terminated with 10 μl of 2.8 M NaOH. The fluorometer utilizedfilters for excitation at 360 nm and emission at 425 nm (e.g., PackardFluorocount fluorometer and PLATE READER Version 3.0 software). Nitritestandards were made with the same experimental media, phenol red freeMEM with 1% BSA+9.5% dextran, in the range 60 nM-8 μM. The NOconcentration range was ˜0.5-3 μM.

A two-factor analysis of variance model was used with the Tukey methodon a 95% confidence interval. The standardized residuals and normalprobability plot of residuals satisfied model requirements for linearity(e.g., statistics software from MINITAB™).

Embodiments of the systems 1, 1101 can include the steady flow componententering (upstream) the test section where the upstream, downstream, andexternal pressures are modulated to impose an oscillatory component onthe steady flow component that resulted in controlled pulsatileconditions, as shown in FIGS. 10A and 10B. As discussed above, byappropriate control of these three pressures (types of dynamic conditionshown in FIG. 17), a wide variety of classes of dynamic conditions (herehemodynamic conditions) can be simulated. FIGS. 10A and 10B show flow,pressure and diameter variation, and 0 deg and −180 deg SPA, which maybe referred to as normal and pathologic hemodynamics from time to tune.In this study, specimen holder 10 was multiplexed to accommodate sixtubes with individual media lines each including real-time monitoringand visualization of flow, pressure, and diameter waveforms therein viaa data acquisition system and software. Flow measurement utilized anoninvasive Doppler ultrasound probe and flow meter (flow meter modelT110 from Transonics™). Pressure measurement was via an invasivecatheter pressure sensor (MPC-500 from Millar®). Noninvasive innerdiameter monitoring required an ultrasound system that utilized a 10 MHztransducer, pulser/receiver, and 50 MHz high-frequency data acquisitioncard (compulite 1250 from GAGE™ Applied Technologies). Sensor signalswere acquired in real time with custom data acquisition software writtenin LABVIEW® and utilized a DAQ card (400 kHz, PCl-6024E from NationalInstruments™). Waveform data was analyzed for desired time periods(e.g., 1 min) and an FFT analysis was performed to determine functionssuch as waveform phase angle differences, magnitude and frequency,calibration scaling, peak max/min, autoscale, sample acquisition rate.Time lags between DAQ cards, sensors, CPU/BUS, and software wereassessed via an external function generator. The flow, pressure, anddiameter measurements were calibrated from the mass flow rate, apneumatic transducer tester (DPM-1B, BIO-TEK® Instruments), and aprecision fabricated tube. All sensors were robust except for thepressure sensor that would require calibration prior to each experiment.

PO₂/PCO₂ control was necessary to ensure proper pH and gasconcentrations for biological experiments. A pH system accommodated sixpH probes (e.g., one per tube) that are multiplexed with a pH meter.PO₂/PCO₂ was measured with a blood gas analyzer (CDI300 blood/gasanalyzer from Terumo). Temperature was controlled at 37° C. via a hotplate and large water bath that was enclosed in a thermal hood. Cellviability was assessed from direct microscope visualization through anintact tube as well as en face staining (slicing the tube open).

The tubular structures 1112 required characteristics of noncytotoxicity,optical transparency for microscope visualization, and mechanicalproperties (e.g., verified using longitudinal stiffness, K_(L), whereK_(L)/A=ΔF/ΔL/L/A:F is force, A is cross-sectional area, and L islength) allowing physiologic diameter variation (±4%) under pressures of70±20 mmHg. The silicone elastomer (Sylgard® 184, Dow Corning) was usedto fabricate the tubes or tubular structures 1112, which in this casewere six cubes of 8 mm inner diameter×15 cm length and wall thicknessesof 500 μm.

Results

One embodiment of the controller 1103 can produce time varying controlsignals f_(j)(t) (e.g., 0 deg SPA and −180 deg SPA) based on inputinformation f_(j)(t) including specimen size, fluid moving capacity(e.g., pump size) and location, and desired dynamic conditions at oralong region A or specimen 12 for pressure flow loop subsystem 1105components. One exemplary method (e.g., controller 1103) will now bedescribed.

In this experiment, theoretical approaches for WSS characterization instraight elastic tubes used sinusoids to approximate prominentcharacteristics of physiologic waveforms and to allow emphasis on theSPA. Alternatively, in vivo measurements of WSS distribution can beused. See, for example, Shung, K. K., Smith, M. B., and Tsui, B. M. W.,1992, Principles of Medical Imaging, Academic, San Diego, the contentsof which are incorporated herein by reference. Note that physiologicwaveforms with multiple harmonics cannot be characterized by a singlevalue of SPA. The calculation of WSS for pulsatile flow in a rigid tubeis known as Womersley's solution. The wall motion in an elastic tubeimposes a radial convective component that affects the WSS. Thenonlinear, elastic tube problem was solved by a perturbation techniquethat produced correction factors for Womersley's solution. The correctedpulsatile WSS component is then added to the steady flow WSS componentthat can be determined from a correction factor applied to Poiseuilleflow. See, for example, Womersley, J. R., 1955, “Method for Calculationof Velocity, Rate of Flow and Viscous Drag in Arteries When the PressureGradlent is Known,” J. Physiol. (London), 127, pp. 553-563; Wang, D. M.,and Tarbell, J. M., 1992, “Nonlinear Analysis of Flow in an Elastic Tube(Artery): Steady Streaming Effects,” J. Fluid Mech., 239, pp. 341-358;Wang, D. M., and Tarbell, J. M., 1995, “Nonlinear Analysis ofOscillatory Flow, With a Nonzero Mean, in an Elastic Tube (Artery),” J.Biomech. Eng., 117(1), pp. 127-135, the contents of which areincorporated herein by reference.

Thus, the WSS solution depends on the phase angle (SPA), α, and the Q,P, and D (e.g., CS) waveforms, which can be provided as inputinformation f_(j)(t) to controller 1103, which then can determine timevarying control signals f_(j)(t) for a selected embodiment of pressureflow loop subsystem 1105 components as described below. The waveformsare decomposed into mean and oscillatory components, where meancomponents are defined with a single overbar and oscillatory(sinusoidal) components are defined with a double overba

WSS= WSS± WSS   (1)

Q= Q± Q   (2)

D= D± D= D±ε  (3)

WSS= WSS _(pois)( Q, D )· C ( Q,ε,φ± WSS _(worm)( Q, D )· C ( Q, Q,ε,φ)  (4)

where WSS _(pois) is the mean WSS determined from Poiseuille flow; WSS_(worm) is the oscillatory WSS determined from Womersley's solution; Cis the correction factor for the mean component [37]; C is thecorrection factor for the oscillatory component [38]; ε is the amplitudeof diameter variation; φ is the SPA. The terms in Eq. (4) are functionsof the parameters in parentheses [i.e., for WSS _(pois)( Q, D), WSS_(pois) is a function Q and D]. The correction factors for theexperimental conditions shown in FIG. 3(A) (0 deg) and FIG. 3(B) (−180deg) are

C (500,0.04,0 deg)=0.993,

C (500,0.04,−180 deg)=1.007,

C (500,700.0.04,0 deg)=0.83,

C (500,700.0.04,−180 deg)=1.23

The resulting WSS waveforms are WSS=10±10 dyne/cm² for both cases. Notethat the correction factors for these experimental conditions indicatethat for SPA=−180 deg, the flow amplitude, Q, should be 23% lower thanthe Womersley flow amplitude, and at SPA=0 deg, Q should be 17% largerthan the Womersley flow amplitude. The correction factors for the meancomponents were negligible for these conditions.

Feedback information FB_(j)(t) can be determined in the selectedembodiments of pressure flow loop subsystem 1105 components and outputto controller 1103 to assess conditions in system 1101 (e.g., at regionA or along conduit 3701) In this study, FB_(j)(t) included at leastQ(t), D(t), P(t), pH, temperature, viability and NO, WSS, CS, SPA.

In this study, long-term stability of the system under pulsatileconditions was assessed via continuous monitoring of the Q, P, and Vwaveforms over a 36 hr period at 37 deg, which showed controlledmaintenance of the dynamic conditions or waveforms. PO₂/PCO₂concentrations were measured after pulsatile conditions with a blood gasanalyzer and were shown to have similar values to incubator controls ofthe same time duration of 17 h, PO₂=141 mmHg and PCO₂=40 mmHg. Thetemperature was very stable (±0.5° C.) and was not affected byopening/closing the thermal hood door. In this study, there were minorvariations in the operating conditions over the 15 cm tube length in thetest section. The variations across the tube length (L) during pulsatileflow at SPA=0 deg and −180 deg were: ΔP=1.5-2 mmHg, ΔQ=10 ml/min, Δτ=0.2dyne/cm² (calculated), and ΔSPA=26 deg.

FIG. 50 shows production of NO from BAECs exposed to hemodynamicconditions in media at 5 hr. In FIG. 50, pairwise significantdifferences indicated by * for 0 deg SPA and −180 deg SPA, # for 0 degSPA and steady state (SS), and ** for dynamic and static controls with pvalues <0.05 (n=5). The biological results in this study depicted inFIG. 46 show a significant decrease in NO quantity for the pathologic−180 deg SPA versus the normal 0 deg SPA case (p<0.05). The 0 deg SPAcase was significantly higher than the steady shear (SS) case (p<0.05).All the dynamic conditions were significantly higher than the staticcases, static control (SC), and pressurized control (PC) (p<0.05). TheSS case verified that the endothelial cells exhibited the anticipatedincreased NO shear response compared to the SC case. The PC case was notsignificantly greater than the SC case.

Systems 1, 1101 simulate normal and pathologic hemodynamics (e.g., FIG.46). Complex physiologic hemodynamic features associated with differentvascular beds can be simulated in vitro. In this embodiment the systemutilized three-dimensional geometries (i.e., silicone tubes) to providea physiologic environment to control or systematically evaluate or modelconcomitant influences of Q, P, and D waveforms on vascular physiologyand/or pathology (e.g., fluid molecules such as gene and proteinexpression profiles).

As shown in FIG. 50, the pressurized control (PC) case was notsignificantly increased compared to the static control (SC) case,implying that the mean pressure and circumferential strain (CS) do norhave a significant influence on NO production compared to steady shearstress. However, concomitant SS and CS affected the NO response ofendothelial cells, modulated by the SPA. The significantly lower NOresponse of the −180 deg versus the 0 deg SPA case along with companioncontrols indicated that the large negative SPA had a negative orpathologic effect on the NO response. Regions of the circulation proneto pathologic development (i.e., atherosclerosis and intimalhyperplasia), such as the aortic abdominal bifurcation and curvedcoronary artery, experience a highly negative SPA. See, for example,Lee, C. S., and Tarbell, J. M., 1997, “Wall Shear Rate Distribution inan Abdominal Aortic Bifurcation Model: Effects of Vessel Compliance andPhase Angle Between Pressure and Flow Waveforms,” J. Biomech. Eng., 119(3), pp. 333-342; and Qiu, Y., and Tarbell, J. M., 2000, “NumericalSimulation of Pulsatile Flow in a Compliant Curved Tube Model of aCoronary Artery,” J. Biomech. Eng., 122(1), pp. 77-85; the contents ofwhich are incorporated herein by reference.

Although embodiments of the invention have been described with referenceto a number of illustrative embodiments thereof, it should be understoodthat numerous other modifications and embodiments can be devised bythose skilled in the art that will fall within the spirit and scope ofthe principles of this invention. More particularly, reasonablevariations and modifications are possible in the component parts and/orarrangements of the subject combination arrangement within the scope ofthe foregoing disclosure, the drawings and the appended claims withoutdeparting from the spirit of the invention. In addition to variationsand modifications in the component parts and/or arrangements,alternative uses will also be apparent to those skilled in the art.

1. A method of conditioning a hybrid synthetic tubular structure havingstem cells and/or endothelial cells on at least one surface to yield afunctional human hybrid hemodialysis access graft, said methodcomprising placing, said hybrid synthetic tubular structure in a systemcapable of producing three dimensional dynamic conditions for asufficient time to yield said functional human hybrid hemodialysisaccess graft.
 2. The method of claim 1, wherein said at least onesurface comprises an inner surface of said synthetic tubular structure.3. The method of claim 1, wherein said at least one surface comprises anouter surface of said synthetic tubular structure.
 4. The method ofclaim 1, wherein said stem cells and/or endothelial cells cover at least50% of said at least one surface of said synthetic tubular structure. 5.The method of claim 1, wherein said stem cells and/or endothelial cellscover at least 60% of said at least one surface of said synthetictubular structure.
 6. The method of claim 5, wherein said at least onesurface comprises an inner surface of said synthetic tubular structure.7. The method of claim 5, wherein said at least one surface comprises anouter surface of said synthetic tubular structure.
 8. The method ofclaim 1, wherein said stem cells and/or endothelial cells cover at least70% of said at least one surface of said synthetic tubular structure. 9.The method of claim 8, wherein said at least one surface comprises aninner surface of said synthetic tubular structure.
 10. The method ofclaim 8, wherein said at least one surface comprises an outer surface ofsaid synthetic tubular structure.
 11. The method of claim 1, whereinsaid stem cells and/or endothelial cells cover at least 80% of said atleast one surface of said synthetic tubular structure.
 12. The method ofclaim 11, wherein said at least one surface comprises an inner surfaceof said synthetic tubular structure.
 13. The method of claim 11, whereinsaid at least one surface comprises an outer surface of said synthetictubular structure.
 14. The method of claim 1, wherein said stem cellsand/or endothelial cells cover at least 90% of said at least one surfaceof said synthetic tubular structure.
 15. The method of claim 14, whereinsaid at least one surface comprises an inner surface of said synthetictubular structure.
 16. The method of claim 14, wherein said at least onesurface comprises an outer surface of said synthetic tubular structure.18. The method of claim 1, wherein said stem cells and/or endothelialcells cover ¹⁰⁰% of said at least one surface of said synthetic tubularstructure.
 19. The method of claim 18, wherein said at least one surfacecomprises an inner surface of said synthetic tubular structure.
 20. Themethod of claim 18, wherein said at least one surface comprises an outersurface of said synthetic tubular structure.